HIGH CONCENTRATION ANTI-TNFalpha ANTIBODY FORMULATION

ABSTRACT

The invention provides a liquid aqueous pharmaceutical formulation comprising a human anti-TNFa antibody, or antigen-binding portion thereof, which reduces pain associated with injection in a subject by at least about 50% when compared to injecting an otherwise identical formulation comprising at least one salt and/or at least one buffer. The invention also provides a liquid aqueous pharmaceutical formulation comprising a human anti-TNFa antibody, or antigen-binding portion thereof, having increased bioavailability upon subcutaneous administration into a subject. The formulation may comprise a therapeutic protein, such as a human anti-TNF-alpha antibody, or an antigen-binding portion thereof, or a biosimilar thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/473,775, filed on Aug. 29, 2014, which is a continuation of U.S.patent application Ser. No. 13/294,692, now U.S. Pat. No. 8,821,865,issued on Sep. 2, 2014, which claims priority to U.S. ProvisionalApplication No. 61/412,728 filed on Nov. 11, 2010 and U.S. ProvisionalApplication No. 61/413,960 filed on Nov. 15, 2010. The entire contentsof each of the foregoing applications are incorporated herein by thisreference in their entireties.

BACKGROUND OF THE INVENTION

The formulation of therapeutic proteins, such as antibodies, is often achallenge given the numerous desirable properties that the formulationmust have to be economically and therapeutically successful, e.g.,stability, suitability for administration, concentration. Duringmanufacturing, storage, and delivery, therapeutic proteins have beenknown to undergo physical and chemical degradations. These instabilitiescan reduce the potency of the protein and increase the risk of adverseevents in patients, and, therefore, significantly impact regulatoryapproval (see, e.g., Wang et al. J. Pharm. Sci. 96:1, 2007). As such, astable protein formulation is essential to the success of a therapeuticprotein.

To be effective, many therapeutic proteins require the administration ofhigh doses, which, ideally, are formulated in high concentrationformulations. High protein concentration formulations are desirable asthey can impact the mode (e.g., intravenous vs. subcutaneous) andfrequency of administration of the drug to a subject.

Despite the benefits of high protein concentration formulations,formulating high concentration therapeutic proteins presents numerouschallenges. For example, increasing protein concentration oftennegatively impacts protein aggregation, solubility, stability, andviscosity (see, e.g., Shire et al. J. Pharm. Sci. 93:1390, 2004).Increased viscosity, which is a very common challenge for high proteinsolutions, can have negative ramifications on administration of theformulation, e.g., felt pain and burning syndromes and limitations inmanufacturing, processing, fill-finish and drug delivery device options(see, e.g., Shire et al. J. Pharm. Sci. 93:1390, 2004). Even fortherapeutic proteins having common structural features, e.g.,antibodies, approved formulations to date have had varying ingredientsand ranges of concentrations. For example, the anti-CD20 antibodyRituxan is formulated for intravenous administration at a concentrationof 10 mg/mL, while the anti-RSV antibody Synagis is formulated forintramuscular administration at a concentration of 100 mg/mL. Thus, highprotein formulations, especially antibody formulations, which can beused for therapeutic purposes remain a challenge.

Another challenge associated with therapeutic proteins, such asantibodies, is drug delivery. While self-administering devices allowpatients to avoid unnecessary trips to medical facilities to receivetreatments, patients' self-awareness and fear of the pain associatedwith self-administration may frequently impact self-administered drugdelivery. Moreover, formulations having high concentrations of proteinmay have high viscosity resulting in increased pain upon delivery,particularly for subcutaneous administration. Thus, there is especiallya need for high concentration formulations that reduce pain associatedwith drug delivery (e.g., self-injection).

Accordingly, there is a need for stable, high concentration proteinformulations that provide dosing and administrative advantages,particularly with respect to a decrease in pain for the patient and/orimproved bioavailability.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnew high-concentration formulations for therapeutic antibodies(including human anti-TNF-α antibodies, or antigen-binding fragmentsthereof, e.g., adalimumab). The formulations of the invention provide anumber of surprising characteristics given the high concentration of thetherapeutic antibody. Specifically, the present invention providespharmaceutical formulations comprising human anti-TNFα antibodies whichsurprisingly have improved bioavailability or decreased pain uponsubcutaneous injection.

In particular, the present invention is based, at least partly, on theunexpected and surprising discovery that a formulation having a highantibody concentration, a surfactant, and a polyol, providesdramatically reduced pain to the patient during drug delivery,particularly subcutaneous administration of the antibody through, forexample, self-injection. The formulations of the invention areestablished, at least in part, on the surprising finding that atherapeutic protein (e.g., an anti-TNF-alpha antibody, orantigen-binding portion thereof), can remain soluble at a high proteinconcentration (e.g., at least about 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 96, 100, 105, 110 mg/ml, or more) and maintain a viscositysuitable for injection (e.g., subcutaneous administration). Theformulation of the present invention is further surprising, in that theformulation does not contain a buffer or a salt, yet has a highconcentration of antibody. Notably, the formulation of the inventionreduces pain associated with injection in a patient by at least about50% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or more)when compared to injecting an otherwise identical formulation comprisingat least one salt and/or at least one buffer.

Thus, in one aspect, the invention provides a liquid aqueous formulationcomprising an anti-TNFα antibody, or antigen-binding portion thereof; asurfactant; and, a polyol; wherein the formulation does not contain abuffer or a salt, and reduces pain associated with injection in apatient by at least about 50% (e.g., at least about 60%, 65%, 70%, 75%,80%, 85%, 90% or more) when compared to injecting an otherwise identicalformulation comprising at least one salt and/or at least one buffer.

In another aspect, the invention provides a liquid aqueous formulationcomprising an isolated human anti-TNFα antibody, or an antigen-bindingportion thereof, a surfactant, and less than 50 mg/mL of a polyol,wherein injection of the formulation into a human subject results in aPain Visual Analog Scale (VAS) score of less than 1.0. In oneembodiment, the invention provides a liquid aqueous formulationconsisting essentially of an isolated human anti-TNFα antibody, or anantigen-binding portion thereof, a surfactant, and less than 50 mg/mL ofa polyol, wherein injection of the formulation into a human subjectresults in a Pain Visual Analog Scale (VAS) score of less than 1.0. Inone embodiment, the VAS scale is from 0 (no pain) to 10 (excruciatingpain)

In a further aspect, the invention provides a liquid aqueous formulationcomprising an isolated human anti-TNFα antibody, or an antigen-bindingportion thereof, a surfactant, and less than 50 mg/ml of a polyol,wherein the formulation does not contain a buffer and a salt, andwherein injection of the formulation reduces pain associated with theinjection in a human subject by at least about 50% when compared toinjection of an otherwise identical formulation that comprises a saltand/or a buffer. In one embodiment, the otherwise identical comprises acitrate and phosphate buffer and sodium chloride.

The invention further provides a liquid aqueous formulation comprisingan anti-TNFα antibody or antigen-binding portion thereof, at aconcentration of at least about 50 mg/mL; a surfactant; and, a polyol,wherein the formulation has a conductivity of less than about 2 mS/cm.In one embodiment, the formulation has a conductivity of less than 1mS/cm. In another embodiment, the formulation has a conductivity of lessthan 0.9 mS/cm.

The invention also provides, in another embodiment, a liquid aqueousformulation comprising an anti-TNFα antibody or antigen-binding portionthereof, at a concentration of at least about 50 mg/mL; a surfactant;and, a polyol, wherein the antibody, or antigen-binding portion thereof,has a hydrodynamic diameter of less than 4 nm in the formulation. In oneembodiment, the antibody or antigen-binding portion thereof, has ahydrodynamic diameter of less than 3 nm in the formulation.

The present invention also provides a liquid aqueous formulationcomprising an isolated human anti-TNFα antibody, or an antigen-bindingportion thereof; a surfactant; and, less than 50 mg/ml of a polyol;wherein the formulation has a characteristic selected from the groupconsisting of a conductivity of less than about 2 mS/cm; a hydrodynamicdiameter (D_(h)) which is at least about 50% less than the D_(h) of theprotein in a buffered solution at a given concentration; and ahydrodynamic diameter (D_(h)) of less than about 4 nm. In oneembodiment, the formulation has a conductivity of less than about 1mS/cm. In another embodiment, the formulation has a conductivity of lessthan about 0.9 mS/cm. In one embodiment, the antibody or antigen-bindingportion thereof, has a hydrodynamic diameter of less than about 3 nm inthe formulation. In another embodiment, the antibody or antigen-bindingportion thereof, has a hydrodynamic diameter of less than about 2 nm inthe formulation.

The invention also provides a liquid aqueous formulation consistingessentially of an anti-TNFα antibody or antigen-binding portion thereof;a surfactant; and, a polyol; wherein the concentration of the anti-TNFαantibody or antigen-binding portion thereof is at least about 50 mg/mL,75 mg/mL, 100 mg/mL, or greater than 100 mg/mL.

In a particular embodiment, the invention provides a liquid aqueousformulation consisting essentially of a concentration of 90-110 mg/ml ofan isolated human anti-TNFα antibody, or an antigen-binding portionthereof, having a light chain variable region (LCVR) having a CDR3domain comprising the amino acid sequence of SEQ ID NO: 3, or modifiedfrom SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5,7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:5, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7;and having a heavy chain variable region (HCVR) having a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8,9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ IDNO: 6, and a CDR1 domain comprising the amino acid sequence of SEQ IDNO: 8; a polysorbate, e.g., polysorbate 80; and, about 38-46 mg/ml of apolyol, e.g., mannitol.

In another aspect, the present invention provides a liquid aqueousformulation comprising an isolated human anti-TNFα antibody, or anantigen-binding portion thereof; a surfactant; and, less than 50 mg/mlof a polyol; wherein the formulation is stable up to about 30 degrees C.for at least about 6 days, about 10, days, or about 14 days, or isstable at about 28 degrees C. for up to about 24 months.

In another aspect, the invention provides a method of administering anisolated human anti-TNFα antibody, or an antigen-binding portionthereof, to a subject such that injection pain is reduced uponadministration, said method comprising subcutaneously administering tothe subject a formulation comprising the antibody, or antigen-bindingportion thereof, such that injection pain is reduced uponadministration, wherein the formulation comprises more than 50 mg/ml ofthe antibody, or antigen-binding portion thereof; a surfactant; and lessthan 50 mg/ml of a polyol. In one embodiment, the injection pain isdetermined to be less than 1.0 according to a Pain Visual Analog Scale(VAS).

In certain embodiments, pain associated with injection is assessed usinga pain visual analog scale (VAS). In one embodiment, the VAS scale isfrom 0 (no pain) to 10 (excruciating pain)

In certain embodiments, the pain associated with injection is assessedafter injection (e.g., immediately, no more than 1, 2, 3, 4, 5, 6, 7, 8,9, 10 minutes, or no more than 15 minutes after injection).

In certain embodiments, the formulation reduces pain associated withinjection in the patient by at least about 60%, 70%, 80% or more, whencompared to injecting the otherwise identical formulation comprising theat least one salt and/or at least one buffer.

The invention further provides a liquid aqueous formulation comprisingan anti-TNFα antibody or antigen-binding portion thereof, at aconcentration of at least about 50, 75, 100 mg/mL, or greater than 100mg/mL; a surfactant; and, a polyol; wherein the formulation does notcontain a buffer and a salt.

In another aspect, the invention provides a liquid aqueous formulationcomprising an isolated human anti-TNFα antibody, or an antigen-bindingportion thereof; a surfactant; and, less than 50 mg/ml of a polyol;wherein the formulation is stable for up to about 30 degrees C. for atleast about 6 days. In one embodiment, the formulation is stable at roomtemperature for at least about 7 days. In one embodiment, theformulation is stable at room temperature for at least about 8 days. Inone embodiment, the formulation is stable at room temperature for atleast about 9 days. In one embodiment, the formulation is stable at roomtemperature for at least about 10 days. In one embodiment, theformulation is stable at room temperature for at least about 11 days. Inone embodiment, the formulation is stable at room temperature for atleast about 12 days. In one embodiment, the formulation is stable atroom temperature for at least about 13 days. In one embodiment, theformulation is stable at room temperature for at least about 14 days. Inone embodiment, the formulation is stable at room temperature for atleast about 15 days.

In one embodiment, the polyol used in the formulation of the inventionis mannitol or sorbitol.

In one embodiment, the formulation of the invention contains about 20-60mg/mL mannitol, or, alternatively, about 30-50 mg/mL. In one embodiment,the formulation contains about 38-46 mg/ml of mannitol.

The present invention is also based, at least in part, on the unexpectedand surprising discovery that a formulation having a high antibodyconcentration and a surfactant provides notably higher bioavailabilitythan similar formulations containing additional excipients, such as abuffer, a polyol and/or a salt.

Thus, in one aspect, the invention provides a liquid aqueous formulationcomprising a surfactant and 30-90 mg of an isolated human anti-TNFαantibody or antigen-binding portion, wherein the formulation has anantibody concentration of 90-110 mg/ml, and wherein the formulationprovides increased bioavailability of the antibody, or antigen-bindingportion thereof, to a human subject upon subcutaneous injection of theformulation relative to a formulation comprising a citrate phosphatebuffer, sodium chloride, and mannitol.

In one aspect, the invention provides a liquid aqueous formulationconsisting essentially of a surfactant and 30-90 mg of an isolated humananti-TNFα antibody or antigen-binding portion, wherein the concentrationof the antibody, or antigen-binding portion thereof, is 90-110 mg/ml.

In another aspect, the invention provides a liquid aqueous formulationcomprising a surfactant and 30-90 mg of an isolated human anti-TNFαantibody, or an antigen-binding portion, wherein the formulation has anantibody concentration of 90-110 mg/ml, and wherein the formulationprovides increased bioavailability of the antibody, or antigen-bindingportion thereof, in a human subject upon subcutaneous injection of theformulation, such that the antibody or antigen-binding portion thereof,has an AUC₀₋₃₆₀ greater than about 1300 μg*hr/ml.

In another aspect, the invention provides a method for improving thebioavailability of an isolated human anti-TNFα antibody, or anantigen-binding portion thereof, in a human subject, said methodcomprising administering a formulation comprising an effective amount ofthe antibody, or antigen-binding portion thereof, and a surfactant tothe subject such that the bioavailability of the antibody, orantigen-binding portion thereof, is improved, wherein the formulationdoes not contain a buffer, a polyol, or a salt.

In a further aspect, the invention provides a method of improving thebioavailability of an isolated human anti-TNFα antibody, or anantigen-binding portion thereof, in a subject, said method comprisingadministering a formulation comprising an effective amount of theantibody, or antigen-binding portion thereof, and a surfactant to thesubject such that the bioavailability of the antibody, orantigen-binding portion thereof, in the subject is improved, at leastabout 15% over a second formulation, wherein the formulation does notcontain a buffer, a polyol, or a salt, and wherein the secondformulation comprises a buffer, a polyol, and a salt. In one embodiment,the bioavailability of the antibody, or antigen-binding portion thereof,is improved at least about 30% over the second formulation. In oneembodiment, the bioavailability of the antibody, or antigen-bindingportion thereof, is improved at least about 40% over the secondformulation.

The invention further provides a method of improving the bioavailabilityof an isolated human anti-TNFα antibody, or an antigen-binding portionthereof, in a human subject, said method comprising administering aformulation comprising a surfactant and an effective amount of theantibody, or antigen-binding portion thereof, to the subject such thatthe bioavailability of the antibody, or antigen-binding portion thereof,is improved, wherein the formulation has a characteristic selected fromthe group consisting of a conductivity of less than about 2 mS/cm; theantibody, or antigen-binding portion thereof, has a hydrodynamicdiameter (D_(h)) which is at least about 50% less than the D_(h) of theantibody, or antigen-binding portion thereof, in a buffered solution atthe given concentration; and the antibody, or antigen-binding portionthereof, has a hydrodynamic diameter (D_(h)) of less than about 4 nm. Inone embodiment, the formulation has a conductivity of less than about 1mS/cm. In another embodiment, the formulation has a conductivity of lessthan about 0.9 mS/cm. In one embodiment, the antibody or antigen-bindingportion thereof, has a hydrodynamic diameter of less than about 3 nm inthe formulation.

In one embodiment, the bioavailability is determined according to eitheran AUC level or a Cmax. In one embodiment, the bioavailability isdetermined according to either an AUC₀₋₃₆₀ or an AUC₀₋₁₃₄₄. In oneembodiment, the bioavailability of the antibody, or antigen-bindingportion thereof, is an AUC₀₋₃₆₀ greater than about 1300 μg*hr/ml whensubcutaneously injected into the human subject.

In certain embodiments, the anti-TNFα antibody is an isolated humanantibody (e.g., a human IgG1 kappa antibody), a humanized antibody, achimeric antibody, or a murine antibody. For example, the chimericantibody may be infliximab or a biosimilar thereof, and the humanantibody may be golimumab or adalimumab, or a biosimilar thereof.

In one embodiment, the human anti-TNFα antibody, or an antigen-bindingportion thereof, is an IgG1 or an IgG4.

In one embodiment, human anti-TNFα antibody, or an antigen-bindingportion thereof, dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M orless and has a k_(off) rate constant of 1×10⁻³ s⁻¹ or less, bothdetermined by surface plasmon resonance. In certain embodiments, thehuman anti-TNFα antibody, or an antigen-binding portion thereof,dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M or less and ak_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determined by surfaceplasmon resonance, and neutralizes human TNFα cytotoxicity in a standardin vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less.

In certain embodiments, the human anti-TNFα antibody, or anantigen-binding portion thereof, has the following characteristics:dissociates from human TNFα with a k_(off) rate constant of 1×10⁻³ s⁻¹or less, as determined by surface plasmon resonance; has a light chainCDR3 domain comprising the ammo acid sequence of SEQ ID NO: 3, ormodified from SEQ ID NO: 3 by a single alanine substitution at position1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutionsat positions 1, 3, 4, 6, 7, 8 and/or 9; and, (c) has a heavy chain CDR3domain comprising the amino acid sequence of SEQ ID NO: 4, or modifiedfrom SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4,5, 6, 8, 9, 10 or 11 or by one to five conservative amino acidsubstitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.

In certain embodiments, the human anti-TNFα antibody, or anantigen-binding portion thereof, has a light chain variable region(LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution atposition 1, 4, 5, 7 or 8, and with a heavy chain variable region (HCVR)having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4,or modified from SEQ ID NO: 4 by a single alanine substitution atposition 2, 3, 4, 5, 6, 8, 9, 10 or 11.

In certain embodiments, the human anti-TNFα antibody, or anantigen-binding portion thereof, has a light chain variable region(LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution atposition 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 5, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 7; and has a heavy chain variable region (HCVR)having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4,or modified from SEQ ID NO: 4 by a single alanine substitution atposition 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising theamino acid sequence of SEQ ID NO: 6, and a CDR1 domain comprising theamino acid sequence of SEQ ID NO: 8.

In certain embodiments, the human anti-TNFα antibody, or anantigen-binding portion thereof, has a light chain variable region(LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a heavychain variable region (HCVR) comprising the amino acid sequence of SEQID NO: 2.

In one embodiment, the human anti-TNFα antibody, or an antigen-bindingportion thereof, comprises the CDRs corresponding to adalimumab.

In one embodiment, the human anti-TNFα antibody, or an antigen-bindingportion thereof, is adalimumab or golimumab, or a biosimilar thereof.

In certain embodiments, the concentration of the human anti-TNFαantibody, or antigen-binding portion thereof, in the formulation is atleast about 50 mg/mL, about 75 mg/mL, about 100 mg/mL, or greater than100 mg/mL. In one embodiment, the concentration of the human anti-TNFαantibody, or antigen-binding portion thereof, in the formulation of theinvention is 90-110 mg/ml. In one embodiment, the concentration of thehuman anti-TNFα antibody, or antigen-binding portion thereof, in theformulation of the invention is 95-105 mg/ml. In one embodiment, theformulation comprises more than 75 mg/ml of the antibody, orantigen-binding portion thereof. In one embodiment, the inventionprovides a stable, liquid aqueous formulation comprising a highconcentration, e.g., 75-125 mg/mL, of a human anti-hTNFα antibody.

In certain embodiments, the surfactant used in the formulation of theinvention is a polysorbate. In one embodiment, the concentration ofpolysorbate is about 0.1-1.5 mg/ml, about 0.2-1.4 mg/ml, about 0.3-1.3mg/ml, about 0.4-1.2 mg/ml, about 0.5-1.1 mg/ml, about 0.6-1.0 mg/ml,about 0.6-1.1 mg/ml, about 0.7-1.1 mg/ml, about 0.8-1.1 mg/ml, or about0.9-1.1 mg/ml. In certain embodiments, the polysorbate is at aconcentration of about 0.1-10 mg/mL, about 0.5-5 mg/mL, about 0.1-2mg/mL, or about 1 mg/mL. In one embodiment, the surfactant ispolysorbate 80.

In certain embodiments, the patient is human, or a non-human mammal.

In certain embodiments, the formulation is Formulation 3 or Formulation4 described in the Examples.

In certain embodiments, the otherwise identical formulation is thecommercially available adalimumab formulation containing adalimumab,sodium chloride, monobasic sodium phosphate dihydrate, dibasic sodiumphosphate dihydrate, sodium citrate, citric acid monohydrate, mannitol,polysorbate 80, and water for Injection.

In one embodiment, the otherwise identical formulation contains a bufferand a salt. In certain embodiments, the salt is a neutral salt, or asalt from a base (e.g., NaOH) used for pH adjustment. In certainembodiments, the buffer comprises a phosphate buffer and/or a citratebuffer. For example, the phosphate buffer may contain about 1.35-1.75mg/mL or about 1.50-1.56 mg/mL of Na₂HPO₄.2H₂O, and about 0.75-0.95mg/mL or about 0.83-0.89 mg/mL of NaH₂PO₄.2H₂O). The citrate buffer maycontain about 1.15-1.45 mg/mL or about 1.30-1.31 mg/mL of citricacid.H₂O, and about 0.2-0.4 mg/mL or about 0.30-0.31 mg/mL of sodiumcitrate dehydrate. The at least one salt may be a neutral salt, such asa neutral sodium salt (e.g., NaCl).

In one embodiment, the formulation of the invention is a pharmaceuticalformulation.

In certain embodiments, the formulation of the invention is suitable forsubcutaneous injection. In one embodiment, the formulation of theinvention is suitable for subcutaneous self-administration by a subject.

In certain embodiments, the volume of the aqueous formulation is no morethan 1.5 mL, 1.0 mL, 0.8 mL, 0.5 mL, or 0.4 mL.

In certain embodiments, the formulation comprises a dose of about 30-90mg of the antibody, or antigen binding portion thereof. In oneembodiment, the formulation comprises about 40 mg of the anti-TNFαantibody, or antigen binding portion thereof. In one embodiment, theformulation comprises about 50 mg of the anti-TNFα antibody, or antigenbinding portion thereof. In one embodiment, the formulation comprisesabout 60 mg of the anti-TNFα antibody, or antigen binding portionthereof. In one embodiment, the formulation comprises about 70 mg of theanti-TNFα antibody, or antigen binding portion thereof. In oneembodiment, the formulation comprises about 80 mg of the anti-TNFαantibody, or antigen binding portion thereof. In one embodiment, theformulation comprises about 90 mg of the anti-TNFα antibody, or antigenbinding portion thereof. In one embodiment, the formulation comprises60-85 mg. In another embodiment, the formulation comprises 70-90 mg. Inyet a further embodiment, the formulation contains 30-110 mg. In oneembodiment, the formulation contains 70-110 mg.

Another aspect of the invention provides a pre-filled syringe orautoinjector device, comprising any of the subject formulationsdescribed herein. In certain embodiments, the aqueous formulation storedin the pre-filled syringe or autoinjector device contains about 40 mg ofadalimumab, or biosimilar thereof. In certain embodiments, the aqueousformulation stored in the pre-filled syringe or autoinjector devicecontains about 80 mg of adalimumab, or biosimilar thereof.

Another aspect of the invention provides a method of treating a disorderassociated with detrimental TNFα activity in a patient, comprisingadministering to the patient any one of the formulations describedherein.

In one embodiment, the formulation or method of the invention is used totreat a subject having rheumatoid arthritis. In one embodiment, theformulation or method of the invention is used to treat a subject havingCrohn's disease. In one embodiment, the formulation or method of theinvention is used to treat a subject having psoriatic arthritis. In oneembodiment, the formulation or method of the invention is used to treata subject having psoriasis. In one embodiment, the formulation or methodof the invention is used to treat a subject having juvenile idiopathicarthritis (JIA). In one embodiment, the formulation or method of theinvention is used to treat a subject having ankylosing spondylitis. Inone embodiment, the formulation or method of the invention is used totreat a subject having ulcerative colitis. In one embodiment, theformulation or method of the invention is used to treat a subject havinghidradenitis suppurativa. In one embodiment, the formulation or methodof the invention is used to treat a subject having diabetic retinopathy.In one embodiment, the formulation or method of the invention is used totreat a subject having giant cell arteritis. In one embodiment, theformulation or method of the invention is used to treat a subject havingBehcet's disease. In one embodiment, the formulation or method of theinvention is used to treat a subject having sarcoidosis, e.g. cutaneoussarcoidosis. In one embodiment, the formulation or method of theinvention is used to treat a subject having axial spondyloarthropathy.In one embodiment, the formulation or method of the invention is used totreat a subject having uveitis.

In one embodiment, the formulation is administered to the subjectaccording to a periodicity selected from the group consisting of weekly,biweekly, every three weeks, and monthly. In one embodiment, theformulation of the invention contains 30-90 mg of a human anti-TNFaantibody, or antigen-binding portion thereof, and is administered on abiweekly dosing regimen. In another embodiment, the formulation of theinvention contains 30-90 mg of a human anti-TNFa antibody, orantigen-binding portion thereof, and is administered according to amonthly dosing regimen. In one embodiment, the formulation of theinvention contains 60-85 mg of a human anti-TNFa antibody, orantigen-binding portion thereof, and is administered on a biweeklydosing regimen. In another embodiment, the formulation of the inventioncontains 60-85 mg of a human anti-TNFα antibody, or antigen-bindingportion thereof, and is administered according to a monthly dosingregimen.

In certain embodiments, the administration of the formulation of theinvention to a subject is via self-administration.

It is contemplated that any one embodiment described herein can becombined with one or more other embodiments of the invention, includingembodiments described only under one aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a panel of graphs that show administration of highconcentration formulations 1 (F1) and 2 (F2) resulted in a significantdecrease in pain assessment at all time points after injection(immediately, 15 minutes, and 30 minutes), compared to the othertreatment groups (F4 and the current commercial formulation).

FIG. 2 shows, on a linear scale, the means and standard deviations ofadalimumab serum concentrations over a time period of 56 days followinga single 40 mg SC dose of adalimumab.

FIGS. 3A and 3B are graphs that show the stability of the variousadalimumab formulations assessed by the number of sum aggregates in theformulations (3A) or the sum aggregates (3B) over a range of polysorbateor a range of polyol.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The term “pain associated with injection (in a patient),” as usedherein, refers to the pain associated with the injection of drug intothe patient's or subject's tissue. In one embodiment, the pain isseparate from the pain caused by the injection device (if any), such asthe injection needle stick. In one embodiment, the pain associated withinjection may originate from the drug formulation being injected intopatient's tissue.

The pain associated with injection may be evaluated using a number ofart-recognized means, such as the Pain Visual Analog Scale (VAS). Thepain measurement is, in one embodiment, quantifiable, such that apercentage pain scale reduction/increase can be directly compared usingstatistical methods. For example, when the Pain Visual Analog Scale isused, a numeric pain value (e.g., average±SD) can be assigned to eachtreatment group, such that a percentage increase or reduction can becalculated. In general, a Visual Analogue Scale (VAS) is a measurementinstrument that measures a characteristic or attitude that is believedto range across a continuum of values (see, e.g., Singer and Thods(1998) Academic Emergency Medicine 5:1007). For example, the amount ofpain that a patient feels ranges across a continuum from none (a scoreof, for example, 0) to an extreme amount of pain (a score of, forexample, 10). From the patient's perspective this spectrum appearscontinuous—their pain does not take discrete jumps, as a categorizationof none, mild, moderate and severe would suggest. Operationally, a VASis usually a horizontal line, 100 mm in length, anchored by worddescriptors at each end, such as “no pain” at one end, and “extremepain” (or some variation thereof) on the other end. The patient marks onthe line at a point (for example, a score of 0-10) that they feelrepresents their perception of their current state. The VAS score maydetermined by measuring in millimeters from the left hand end of theline to the point that the patient marks.

There are various ways in which VAS have been presented, includingvertical lines and lines with extra descriptors. See Wewers & Lowe (“Acritical review of visual analogue scales in the measurement of clinicalphenomena.” Research in Nursing and Health 13: 227-236, 1990,incorporated by reference herein) provide an informative discussion ofthe benefits and shortcomings of different styles of VAS.

The term “liquid formulation” refers to a formulation in a liquid stateand is not intended to refer to resuspended lyophilized formulations. Aliquid formulation of the invention is stable upon storage, and does notrely upon lyophilization (or other state change methods, e.g., spraydrying) for stability.

The term “liquid aqueous formulation” refers to a liquid formulationusing water as a solvent. In one embodiment, a liquid aqueousformulation is a formulation that maintains stability (e.g., chemicaland/or physical stability/and/or biological activity) without the needfor lyophilization, spray-drying, and/or freezing.

The term “pharmaceutical,” as used herein, refers to a composition,e.g., an aqueous formulation, that it is useful for treating a diseaseor disorder.

The term “subject” or “patient” is intended to include mammalianorganisms. Examples of subjects/patients include humans and non-humanmammals, e.g., non-human primates, dogs, cows, horses, pigs, sheep,goats, cats, mice, rabbits, rats, and transgenic non-human animals. Inspecific embodiments of the invention, the subject is a human.

The term “excipient” refers to an agent which may be added to aformulation to provide a desired characteristic, e.g., consistency,improving stability, and/or to adjust osmolality. Examples of commonlyused excipients include, but are not limited to, sugars, polyols, aminoacids, surfactants, and polymers.

A commonly used excipient is a polyol. As used herein, a “polyol” is asubstance with multiple hydroxyl groups, and includes sugars (reducingand nonreducing sugars), sugar alcohols and sugar acids. Non-limitingexamples of polyols are fructose, mannose, maltose, lactose, arabinose,xylose, ribose, rhamnose, galactose, glucose, sucrose, trehalose,sorbose, melezitose, raffinose, mannitol, xylitol, erythritol, threitol,sorbitol, glycerol, L-gluconate and metallic salts thereof. In oneembodiment, the polyol used in the formulation or methods of theinvention is mannitol. In one embodiment, the polyol used in theformulation or methods of the invention is sorbitol.

A “therapeutically active antibody” or “therapeutic antibody” refers toan antibody which may be used for therapeutic purposes, i.e., for thetreatment of a disorder in a subject. It should be noted that whiletherapeutic proteins may be used for treatment purposes, the inventionis not limited to such use, as said proteins may also be used for invitro studies.

As used herein, “buffer” is an agent(s) in a solution that allows thesolution to resist changes in pH by the action of its acid-baseconjugate components. Examples of buffers include acetate (e.g. sodiumacetate), succinate (such as sodium succinate), gluconate, histidine,methionine, citrate, phosphate, citrate/phosphate, imidazole,combinations thereof, and other organic acid buffers. In one embodiment,a buffer is not a protein. A buffer may provide a solution with a pH inthe range from about 4 to about 8; from about 4.5 to about 7; or fromabout 5.0 to about 6.5.

Although the formulations of the invention do not contain a buffer(s),otherwise identical formulations containing one or more buffers may beused for pain or bioavailability comparison purposes. Examples of suchbuffers include phosphate, acetate (e.g., sodium acetate), succinate(such as sodium succinate), gluconate, glutamate, histidine, citrate andother organic acid buffers. In one embodiment, a representative bufferin the otherwise identical formulation comprises a citrate buffer and/ora phosphate buffer.

As used herein, the term “surfactant” generally includes an agent thatprotects the protein, e.g., antibody, from air/solutioninterface-induced stresses, solution/surface induced-stresses, to reduceaggregation of the antibody, or to minimize the formation ofparticulates in the formulation. Exemplary surfactants include, but arenot limited to, nonionic surfactants such as polysorbates (e.g.polysorbates 20 and 80) or poloxamers (e.g. poloxamer 188). The term“surfactant” or “detergent” includes nonionic surfactants such as, butnot limited to, polysorbates. In one embodiment, a surfactant includespoloxamers, e.g., Poloxamer 188, Poloxamer 407; polyoxyethylene alkylethers, e.g., Brij 35®, Cremophor A25, Sympatens ALM/230; andpolysorbates/Tweens, e.g., Polysorbate 20 (Tween 20), Polysorbate 80(Tween 80), Mirj, and Poloxamers, e.g., Poloxamer 188.

A “stable” formulation is one in which the antibody therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity during the manufacturing process and/or uponstorage. Various analytical techniques for measuring protein stabilityare available in the art and are reviewed in Peptide and Protein DrugDelivery 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y.,Pubs. (1991); and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90(both incorporated by reference). For example, in one embodiment, thestability of a protein is determined according to the percentage ofmonomer protein in the solution, with a low percentage of degraded(e.g., fragmented) and/or aggregated protein. In one embodiment, theformulation may be stable at room temperature, at about 25-30° C., or at40° C. for at least 1 month and/or stable at about 2-8° C. for at least1 month, 1 year, or, alternatively, for at least 2 years. In anotherembodiment, the formulation may be stable up to about 30 degrees C. forat least about 6 days, about 10, days, or about 14 days, or is stable atabout 28 degrees C. for up to about 24 months. In one embodiment, theformulation may be stable following freezing (to, e.g., −70° C.) andthawing of the formulation, hereinafter referred to as a “freeze/thawcycle.”

An antibody “retains its physical stability” in a pharmaceuticalformulation if it shows substantially no signs of, e.g., aggregation,precipitation and/or denaturation upon visual examination of colorand/or clarity, or as measured by UV light scattering or by sizeexclusion chromatography. Aggregation is a process whereby individualmolecules or complexes associate covalently or non-covalently to formaggregates. Aggregation can proceed to the extent that a visibleprecipitate is formed.

Stability, such as physical stability of a formulation, may be assessedby methods well-known in the art, including measurement of a sample'sapparent attenuation of light (absorbance, or optical density). Such ameasurement of light attenuation relates to the turbidity of aformulation. The turbidity of a formulation is partially an intrinsicproperty of the protein dissolved in solution and is commonly determinedby nephelometry, and measured in Nephelometric Turbidity Units (NTU).

The degree of turbidity, e.g., as a function of the concentration of oneor more of the components in the solution, e.g., protein and/or saltconcentration, is also referred to as the “opalescence” or “opalescentappearance” of a formulation. The degree of turbidity can be calculatedby reference to a standard curve generated using suspensions of knownturbidity. Reference standards for determining the degree of turbidityfor pharmaceutical compositions can be based on the EuropeanPharmacopeia criteria (European Pharmacopoeia, Fourth Ed., Directoratefor the Quality of Medicine of the Council of Europe (EDQM), Strasbourg,France). According to the European Pharmacopeia criteria, a clearsolution is defined as one with a turbidity less than or equal to areference suspension which has a turbidity of approximately 3 accordingto European Pharmacopeia standards. Nephelometric turbidity measurementscan detect Rayleigh scatter, which typically changes linearly withconcentration, in the absence of association or nonideality effects.Other methods for assessing physical stability are well-known in theart.

An antibody “retains its chemical stability” in a pharmaceuticalformulation, if the chemical stability at a given time is such that theantibody is considered to still retain its biological activity asdefined below. Chemical stability can be assessed by, e.g., detectingand quantifying chemically altered forms of the antibody. Chemicalalteration may involve size modification (e.g. clipping) which can beevaluated using size exclusion chromatography, SDS-PAGE and/ormatrix-assisted laser desorption ionization/time-of-flight massspectrometry (MALDI/TOF MS), for example. Other types of chemicalalteration include charge alteration (e.g. occurring as a result ofdeamidation or oxidation) which can be evaluated by ion-exchangechromatography, for example.

An antibody “retains its biological activity” in a pharmaceuticalformulation, if the antibody in a pharmaceutical formulation isbiologically active for its intended purpose. For example, biologicalactivity is retained if the biological activity of the antibody in thepharmaceutical formulation is within about 30%, about 20%, or about 10%(within the errors of the assay) of the biological activity exhibited atthe time the pharmaceutical formulation was prepared (e.g., asdetermined in an antigen binding assay).

In a pharmacological sense, in the context of the present invention, a“therapeutically effective amount” or “effective amount” of an antibodyrefers to an amount effective in the prevention or treatment oralleviation of a symptom of a disorder for the treatment of which theantibody is effective.

The term “human TNF-alpha” (abbreviated herein as hTNF-alpha, TNFα, orsimply hTNF), as used herein, is intended to refer to a human cytokinethat exists as a 17 kDa secreted form and a 26 kDa membrane associatedform, the biologically active form of which is composed of a trimer ofnoncovalently bound 17 kDa molecules. The structure of hTNF-alpha isdescribed further in, for example, Pennica, D., et al. (1984) Nature312:724-729; Davis, J. M., et al. (1987) Biochem 26:1322-1326; andJones, E. Y., et al. (1989) Nature 338:225-228. The term human TNF-alphais intended to include recombinant human TNF-alpha (rhTNF-alpha), whichcan be prepared by standard recombinant expression methods or purchasedcommercially (R & D Systems, Catalog No. 210-TA, Minneapolis, Minn.).

The term “antibody,” as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Other naturally occurring antibodies of altered structure, such as, forexample, camelid antibodies, are also included in this definition. Eachheavy chain is comprised of a heavy chain variable region (abbreviatedherein as HCVR or VH) and a heavy chain constant region. The heavy chainconstant region is comprised of three domains, CH1, CH2 and CH3. Eachlight chain is comprised of a light chain variable region (abbreviatedherein as LCVR or VL) and a light chain constant region. The light chainconstant region is comprised of one domain, CL. The VH and VL regionscan be further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In one embodiment of the invention, the formulation containsan antibody with CDR1, CDR2, and CDR3 sequences like those described inU.S. Pat. Nos. 6,090,382 and 6,258,562, each incorporated by referenceherein. In certain embodiments, the formulation contains an antibody asclaimed in U.S. Pat. Nos. 6,090,382 and 6,258,562.

As used herein, the term “CDR” refers to the complementarity determiningregion within a antibody variable sequence. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the heavy and lightchain variable regions. The exact boundaries of these CDRs have beendefined differently according to different systems. The system describedby Kabat (Id.) not only provides an unambiguous residue numbering systemapplicable to any variable region of an antibody, but also providesprecise residue boundaries defining the three CDRs. These CDRs may bereferred to as Kabat CDRs. Chothia et al. found that certainsub-portions within Kabat CDRs adopt nearly identical peptide backboneconformations, despite having great diversity at the level of amino acidsequence (Chothia et al. (1987) Mol. Biol. 196:901-917; Chothia et al.(1989) Nature 342:877-883) These sub-portions were designated as L1, L2and L3 or H1, H2 and H3 where the “L” and the “H” designates the lightchain and the heavy chains regions, respectively. These regions may bereferred to as Chothia CDRs, which have boundaries that overlap withKabat CDRs. Other boundaries defining CDRs overlapping with the KabatCDRs have been described by Padlan (1995) FASEB J. 9:133-139 andMacCallum (1996) J. Mol. Biol. 262(5):732-45. Still other CDR boundarydefinitions may not strictly follow one of the herein described systems,but will nonetheless overlap with the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, although certainembodiments use Kabat or Chothia defined CDRs. In one embodiment, theantibody used in the methods and compositions of the invention includesthe six CDRs from the antibody adalimumab.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hTNF-alpha). It has been shown that the antigen-binding functionof an antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Inone embodiment of the invention, the formulation contains anantigen-binding portions described in U.S. Pat. Nos. 6,090,382 and6,258,562, each incorporated by reference herein.

The phrase “recombinant antibody” refers to antibodies that areprepared, expressed, created or isolated by recombinant means, such asantibodies expressed using a recombinant expression vector transfectedinto a host cell, antibodies isolated from a recombinant, combinatorialantibody library, antibodies isolated from an animal (e.g., a mouse)that is transgenic for human immunoglobulin genes (see e.g., Taylor etal. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared,expressed, created or isolated by any other means that involves splicingof particular immunoglobulin gene sequences (such as humanimmunoglobulin gene sequences) to other DNA sequences. Examples ofrecombinant antibodies include recombinant human, chimeric, CDR-graftedand humanized antibodies.

The term “human antibody,” as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies used in theinvention may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody,” as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

An “isolated antibody,” as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hTNF-alpha is substantially free of antibodies that specificallybind antigens other than hTNF-alpha). An isolated antibody thatspecifically binds hTNF-alpha may, however, have cross-reactivity toother antigens, such as TNF-alpha molecules from other species.Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals.

A “neutralizing antibody,” as used herein (or an “antibody thatneutralized hTNF-alpha activity”), is intended to refer to an antibodywhose binding to hTNF-alpha results in inhibition of the biologicalactivity of hTNF-alpha. This inhibition of the biological activity ofhTNF-alpha can be assessed by measuring one or more indicators ofhTNF-alpha biological activity, such as hTNF-alpha-induced cytotoxicity(either in vitro or in vivo), hTNF-alpha-induced cellular activation andhTNF-alpha binding to hTNF-alpha receptors. These indicators ofhTNF-alpha biological activity can be assessed by one or more of severalstandard in vitro or in vivo assays known in the art, and described inU.S. Pat. Nos. 6,090,382 and 6,258,562, each incorporated by referenceherein. In one embodiment, the ability of an antibody to neutralizehTNF-alpha activity is assessed by inhibition of hTNF-alpha-inducedcytotoxicity of L929 cells. As an additional or alternative parameter ofhTNF-alpha activity, the ability of an antibody to inhibithTNF-alpha-induced expression of ELAM-1 on HUVEC, as a measure ofhTNF-alpha-induced cellular activation, can be assessed.

The term “surface plasmon resonance,” as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson, U., et al. (1993) Ann Biol. Clin.51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson,B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al.(1991) Anal. Biochem. 198:268-277.

The term “k_(on),” as used herein, is intended to refer to the on rateconstant for association of a binding protein (e.g., an antibody) to theantigen to form the, e.g., antibody/antigen complex as is known in theart.

The term “k_(off),” as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(d),” as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction andrefers to the value obtained in a titration measurement at equilibrium,or by dividing the dissociation rate constant (k_(off)) by theassociation rate constant (k_(on)).

As used herein, “biosimilar” (of an approved referenceproduct/biological drug, such as a protein therapeutic, antibody, etc.)refers to a biologic product that is similar to the reference productbased upon data derived from (a) analytical studies that demonstratethat the biological product is highly similar to the reference productnotwithstanding minor differences in clinically inactive components; (b)animal studies (including the assessment of toxicity); and/or (c) aclinical study or studies (including the assessment of immunogenicityand pharmacokinetics or pharmacodynamics) that are sufficient todemonstrate safety, purity, and potency in one or more appropriateconditions of use for which the reference product is licensed andintended to be used and for which licensure is sought for the biologicalproduct. In one embodiment, the biosimilar biological product andreference product utilize the same mechanism or mechanisms of action forthe condition or conditions of use prescribed, recommended, or suggestedin the proposed labeling, but only to the extent the mechanism ormechanisms of action are known for the reference product. In oneembodiment, the condition or conditions of use prescribed, recommended,or suggested in the labeling proposed for the biological product havebeen previously approved for the reference product. In one embodiment,the route of administration, the dosage form, and/or the strength of thebiological product are the same as those of the reference product. Inone embodiment, the facility in which the biological product ismanufactured, processed, packed, or held meets standards designed toassure that the biological product continues to be safe, pure, andpotent. The reference product may be approved in at least one of theU.S., Europe, or Japan.

The term “dosing”, as used herein, refers to the administration of asubstance (e.g., an anti-TNFa antibody) to achieve a therapeuticobjective (e.g., the treatment of a TNFa-associated disorder).

The terms “weekly dosing regimen”, “weekly dosing” and “weeklyadministration” as used herein, refer to a certain time course (orperiodicity) of administering a substance (e.g., an anti-TNFα antibody)to a subject to achieve a therapeutic objective (e.g., the treatment ofa TNFα.-associated disorder). In one embodiment, the antibody, orantigen-binding portion thereof, is administered every 6-8 days, or,alternatively, every 7 days.

The terms “biweekly dosing regimen”, “biweekly dosing”, and “biweeklyadministration”, as used herein, refer to a certain time course (orperiodicity) of administering a substance (e.g., an anti-TNFα antibody)to a subject to achieve a therapeutic objective (e.g., the treatment ofa TNFα.-associated disorder). The biweekly dosing regimen is notintended to include a weekly dosing regimen. In one embodiment, theantibody, or antigen-binding portion thereof, is administered every 9-19days, more preferably, every 11-17 days, even more preferably, every13-15 days, and most preferably, every 14 days.

The terms “monthly dosing regimen”, “monthly dosing”, and “monthlyadministration”, as used herein, refer to a certain time course (orperiodicity) of administering a substance (e.g., an anti-TNFα antibody)to a subject to achieve a therapeutic objective (e.g., the treatment ofa TNFα.-associated disorder). In one embodiment, a monthly dosingregimen means that the antibody, or antigen-binding portion thereof, isadministered every 28-31 days. In another embodiment, a monthly dosingregimen means that the antibody, or antigen-binding portion thereof, isadministered once a month, e.g. on the same day each month, such as, forexample, the first day of each month.

AUC, Cmax, and Tmax are pharmacokinetic parameters that may be used tocharacterize the pharmacokinetic responses of a particular drug productin an animal or human subject. The term “AUC” refers to the “area underthe curve” that represents changes in blood, serum, or plasmaconcentrations of a substance, e.g., a human anti-TNFα antibody, overtime. As used herein, the term “Cmax” refers to the maximum or peakblood, serum, or plasma concentration of substance observed in a subjectafter its administration. The term “Tmax” refers to the time at whichthe Cmax occurred, as measured from the time point of administration.”

The term “hydrodynamic diameter” or “D_(h)” of a particle refers to thediameter of a sphere that has the density of water and the same velocityas the particle. Thus, the term “hydrodynamic diameter of an antibody”as used herein refers to a size determination for an antibody, or anantigen-binding portion thereof, e.g., a human anti-TNFα antibody, orantigen-binding fragment thereof, in solution using dynamic lightscattering (DLS). A DLS-measuring instrument measures the time-dependentfluctuation in the intensity of light scattered from the antibody, orantigen-binding fragment thereof, in solution at a fixed scatteringangle. D_(h) is determined from the intensity autocorrelation functionof the time-dependent fluctuation in intensity. Scattering intensitydata are processed using DLS instrument software to determine the valuefor the hydrodynamic diameter and the size distribution of thescattering molecules, e.g. the human anti-TNFα antibody, orantigen-binding fragment thereof, specimen.

The term “conductivity,” as used herein, refers to the ability of anaqueous solution to conduct an electric current between two electrodes.Generally, electrical conductivity or specific conductivity is a measureof a material's ability to conduct an electric current. In solution, thecurrent flows by ion transport. Therefore, with an increasing amount ofions present in the aqueous solution, the solution will have a higherconductivity. The unit of measurement for conductivity is mmhos (mS/cm),and can be measured using a conductivity meter sold, e.g., by OrionResearch, Inc. (Beverly, Mass.). The conductivity of a solution may bealtered by changing the concentration of ions therein. For example, theconcentration of buffer and/or salt, in the solution may be altered inorder to achieve the desired conductivity.

Conductivity of a solution is measured according to methods known in theart. Conductivity meters and cells may be used to determine theconductivity of the aqueous formulation, and should be calibrated to astandard solution before use. Examples of conductivity meters availablein the art include MYRON L Digital (Cole Parmer®), Conductometer(Metrohm AG), and Series 3105/3115 Integrated Conductivity Analyzers(Kemotron).

Conductivity measurements may be taken with any commercially availableconductivity meter suitable for conductivity analysis in proteinsolutions, e.g. conductivity meter Model SevenMulti, with expansioncapacity for broad pH range (Mettler Toledo, Schwerzenbach,Switzerland). The instrument is operated according to the manufacturersinstructions (e.g., if the conductivity sensor is changed in the MettlerToledo instrument, calibration must be performed again, as each sensorhas a different cell constant; refer to Operating Instructions of ModelSevenMulti conductivity meter). If the instructions are followed,conductivity measurements can be taken by directly immersing themeasuring probe into the sample solution.

Various aspects of the invention are described in further detail in thefollowing subsections.

II. Formulations and Methods of the Invention

The present invention features stable, liquid aqueous pharmaceuticalformulations comprising an anti-TNFα antibody, or an antigen bindingportion thereof, having improved properties as compared toart-recognized formulations. While high concentration formulationscontaining human anti-TNFα antibodies are known in the art (see, forexample, US20060153846 and US20100278822), the instant inventionprovides high concentration formulations having unexpectedcharacteristics, i.e., significantly decreased pain or increasedbioavailability. The formulations of the invention are based, at leastin part, on the combination of only one or two excipients, i.e., asurfactant and a polyol or, alternatively, a surfactant alone. Despitehaving few excipients, the formulations of the invention contain a highconcentration of an antibody, e.g. 90-110 mg/ml, and are stable.

As described in the working examples below, a formulation containing anantibody concentration of more than 50 mg/ml of an isolated humananti-TNFα antibody, less than 50 mg/ml of a polyol, (such as mannitol),and a surfactant, (such as a polysorbate), was shown to havedramatically reduced pain upon injection relative to other highconcentration formulations, including the commercial adalimumabformulation described in US20060153846, and the formulation described inUS20100278822, each of which is incorporated by reference herein. Thus,in one embodiment, the formulations of the invention are associated witha reduction of pain, despite having a high antibody concentration (e.g.,100 mg/mL) and having no buffer or salt. The low-pain formulationsdescribed herein are based, at least in part, on the surprising findingthat by removing or excluding salt (e.g., NaCl) and/or a buffer (e.g., aphosphate/citrate buffer) the concentration of a human anti-TNF alphaantibody in a formulation can be increased, e.g., to about 100 mg/mL,while decreasing pain upon delivery to a patient.

In one embodiment, the formulation of the invention is surprising, inthat the formulation does not contain a buffer or a salt, and reducespain associated with injection in a patient by at least about 50% whencompared to injecting an otherwise identical formulation comprising atleast one salt and/or at least one buffer. In one embodiment, theformulation reduces pain associated with the injection in a humansubject by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or 80% (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 50, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, or 80%) when compared to the injection of anotherwise identical formulation that further comprises a salt and/or abuffer.

In one embodiment, the otherwise identical formulation used for paincomparison assay comprises at least one buffer, such as a citrate bufferand a phosphate buffer, and/or a salt, e.g., NaCl. For example, thebuffer (excluded from the formulation of the invention and present inthe reference formulation for pain comparisons) may include citric acidmonohydrate, sodium citrate, disodium phosphate dihydrate, and/or sodiumdihydrogen phosphate dihydrate. The buffer may include about 1.15-1.45mg/ml of citric acid (e.g., about 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, or1.45), about 0.2-0.4 mg/mL of sodium citrate dehydrate (e.g., about 0.2,0.25, 0.3, 0.35, or 0.4), about 1.35-1.75 mg/mL of disodium phosphatedehydrate (e.g., about 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70,or 1.75), about 0.75-0.95 mg/mL of sodium dihydrogen phosphate dehydrate(e.g., about 0.75, 0.80, 0.85, 0.9, or 0.95). Values and rangesintermediate to the aforementioned concentrations are also intended tobe part of this invention. In addition, ranges of values using acombination of any of the above-recited values as upper and/or lowerlimits are intended to be included, e.g., 0.1 to 0.5 mg/mL or 1.20-1.40mg/mL. In one embodiment, the pH of the formulation is adjusted withsodium hydroxide.

In one embodiment, the formulation of the invention includes highconcentrations of human anti-TNFa antibodies, or antigen bindingportions thereof, e.g., 90-110 mg/ml, a polyol at a concentration lessthan 50 mg/ml, and a surfactant, such that the formulation is suitablefor administration without significant pain as determined by a visualanalog scale (VAS) score. In one embodiment, the formulation and methodsof the invention include high concentrations of anti-TNFα antibodies, orantigen binding portions thereof, and no buffer or salt, such that theyare suitable for, administration, e.g., subcutaneous administration,without significant felt pain as determined by a visual analog scale(VAS) score. For example, the formulation of the invention may result ina VAS score of less than 1 on a scale of 0 (no pain) to 10 (worstimaginable pain) following subcutaneous injection. As described inExample 1, a formulation having 100 mg/ml of adalimumab, polysorbate 80,and mannitol (less than 50 mg/ml) resulted in a VAS score of less than1, e.g., 0.56, whereas other high antibody concentration formulationsresulted in VAS scores ranging from 1.79 to 4.12.

In one embodiment, the invention provides a liquid aqueous formulationcomprising an isolated human anti-TNFα antibody, or an antigen-bindingportion thereof, a surfactant, and less than 50 mg/ml of a polyol,wherein subcutaneous injection of the formulation results in a PainVisual Analog Scale score of less than 1.0 following injection. In oneembodiment, the formulation does not contain a buffer and a salt, andresults in a reduction of pain of at least about 50% upon subcutaneousinjection when compared to an injection of an otherwise identicalformulation that further comprises a salt and/or a buffer(s).

Thus, in one aspect of the invention, liquid formulations of theinvention have advantageous tolerability properties in that theformulations produce less pain relative to formulations containing abuffer and a salt. In certain embodiments, the formulation reduces painassociated with injection (or any other form of administration) in asubject. In some embodiments, pain associated with injection is reducedby at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, or at leastabout 95% (e.g., at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, or 95%). In one embodiment, pain is reduced by at leastabout 50%.

Pain may be evaluated using any type of pain assessment known in theart, including, for example, visual analog scales, qualitativeassessments of pain, or needle pain assessments. For example,subject-perceived injection site pain may be assessed using the PainVisual Analog Scale (VAS). A VAS is a measurement instrument thatmeasures pain as it ranges across a continuum of values, e.g., from noneto an extreme amount of pain. Operationally a VAS is a horizontal line,about 100 mm in length, anchored by numerical and/or word descriptors,e.g., 0 or 10, or “no pain” or “excruciating pain,” optionally withadditional word or numeric descriptors between the extremes, e.g., mild,moderate, and severe; or 1 through 9) (see, e.g., Lee J S, et al. (2000)Acad Emerg Med 7:550, or Singer and Thods (1998) Academic EmergencyMedicine 5:1007). Pain may be assessed at a single time or at varioustimes following administration of a formulation of the invention suchas, for example, immediately after injection, at about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 minutes after injection.

In a certain embodiment of the invention, injection of the formulationinto a subject results in a Pain Visual Analog Scale score of less than0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, or 5.0 on a scale of 0 (no pain)to 10 (excruciating pain).

Other tools for pain assessment are known in the art, including, forexample, the Numerical Rating Scale, the Verbal Rating Scale, and theBrief Pain Inventory. Such tools could also be used to assess pain inaccordance with the invention.

Additional indices for skin irritation may be used, including, e.g., theDraize Scale (hemorrhage, petechiae, erythema, edema, pruritus).

Formulations of the invention containing a polyol preferably containless than about 50 mg of the polyol. In one embodiment, the formulationscontain less than about 45 mg/mL of the polyol. In another embodiment,the formulations of the invention contain about 38-46 mg/mL of thepolyol (e.g., mannitol), e.g., about 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mg/mL of the polyol.In addition, ranges of values using a combination of any of the aboverecited values as upper and/or lower limits are intended to be included,e.g., 39-45 mg/ml, 40-44 mg/ml, or 37-47 mg/ml. In one embodiment, theformulations of the invention contain about 12-72 mg/ml of polyol, e.g.,mannitol. In one embodiment, suitable polyols for use in theformulations and methods of the invention are mannitol or sorbitol.

In one embodiment, the formulation of the invention contains adalimumab(or a biosimilar thereof), polysorbate 80, mannitol, and water forinjection. In one embodiment, the formulation contains 80 mg ofadalimumab, water for injection, 42 mg/ml of mannitol, and 1 mg/ml ofpolysorbate 80. In one embodiment, the formulation may contain 20-110mg, alternatively 20-90 mg of adalimumab or, alternatively, 30-80 mg ofthe antibody. In one embodiment, the formulation contains 30 mg, 31 mg,32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg 69 mg, 70 mg, 71 mg, 72 mg,73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 101 mg, 102 mg,103 mg, 104 mg, 105 mg, 106 mg, 107 mg, 108 mg, 109 mg, or 110 mg of theantibody. Ranges including the aforementioned numbers are also includedin the invention, e.g., 70-90 mg, 65-95, or 60-85 mg.

The present invention is also based, at least in part on the surprisingdiscovery that a liquid aqueous pharmaceutical formulation having a highconcentration of a human anti-TNFα antibody, or antigen binding portionthereof, and a surfactant (i.e., in the absence of additionalexcipients), has greater bioavailability than other high concentrationformulations having additional excipients. As described in the workingexamples below, a formulation containing more than 50 mg/ml of anisolated human anti-TNFα antibody, and a polysorbate was shown to haveincreased bioavailability relative to other high concentrationformulations, including the commercial adalimumab formulation describedin US20060153846.

As described in Example 2 below, bioavailability of an anti-TNFαantibody can be increased by combining the antibody with a surfactant,e.g., polysorbate 80. The increase in bioavailability is based on thecombination of the antibody and surfactant and the omission or removalof other excipients, including a buffer, polyol, and salt. The increasein bioavailability results in an AUC₀₋₃₆₀ of the anti-TNFα antibody, oran antigen-binding portion thereof, of greater than about 1300 μg*hr/mlor an AUC₀₋₁₃₄₄ of the anti-TNFα antibody, or an antigen-binding portionthereof, of greater than about 2600 μg*hr/ml, when subcutaneouslyinjected into a human subject.

Accordingly, the present invention provides methods for improving thebioavailability of an isolated anti-TNFα antibody, or an antigen-bindingportion thereof, in a pharmaceutical formulation. The methods includecombining a therapeutically effective amount of the anti-TNFα antibody,or antigen-binding portion thereof, with a surfactant and excluding orremoving other excipients, e.g., a buffer(s), salt, and polyol, orcombinations thereof, such that the bioavailability of the antibody, orantigen-binding portion thereof, is improved. In one embodiment, theformulation is injected subcutaneously into a human subject. The methodsmay improve the bioavailability by providing an AUC₀₋₃₆₀ of theanti-TNFα antibody, or an antigen-binding portion thereof, of greaterthan about 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325,1350, 1375, 1400, 1425, 1450, 1475, or about 1500 μg*hr/ml whensubcutaneously injected into a human subject.

The invention further provides a method of improving the bioavailabilityof an isolated human anti-TNFα antibody, or an antigen-binding portionthereof, in a subject, said method comprising administering aformulation comprising a surfactant and an effective amount of theantibody, or antigen-binding portion thereof, to the subject such thatthe bioavailability of the antibody, or antigen-binding portion thereof,in the subject is improved at least about 15% over a second formulation.In one embodiment, the formulation of the invention does not contain abuffer, a polyol, or a salt, and the second formulation comprises abuffer, a polyol, and a salt. In one embodiment, the bioavailability ofthe antibody, or antigen-binding portion thereof, is improved at leastabout 30% over the second formulation. In one embodiment, thebioavailability of the antibody, or antigen-binding portion thereof, isimproved at least about 40% over the second formulation. In oneembodiment, the bioavailability may be determined according to either anAUC level, e.g., AUC₀₋₃₆₀ or an AUC₀₋₁₃₄₄, or a Cmax.

In one embodiment, the present invention provides a liquid aqueousformulation which includes a surfactant and about 30-90 mg of anisolated human anti-TNFα antibody or antigen-binding portion, whereinthe formulation has an antibody concentration of about 90-110 mg/ml, andwherein the formulation provides increased bioavailability of theantibody, or antigen-binding portion thereof, to a human subject uponsubcutaneous injection of the formulation relative to a formulationcomprising citrate phosphate buffer, sodium chloride, and mannitol.

In one embodiment, the present invention provides liquid aqueousformulations which include a surfactant and 30-90 mg of an isolatedhuman anti-TNFα antibody, or an antigen-binding portion, wherein theformulation has an antibody concentration of 90-110 mg/ml, and whereinthe formulation provides increased bioavailability of the antibody, orantigen-binding portion thereof, to a human subject upon subcutaneousinjection of the formulation, such that the antibody or antigen-bindingportion thereof, has an AUC₀₋₃₆₀ greater than about 1100, 1125, 1150,1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450,1475, or about 1500 mg*hr/ml.

In one embodiment, the formulation of the invention contains adalimumab(or a biosimilar thereof), polysorbate 80, and water for injection. Inone embodiment, the formulation contains 80 mg of adalimumab, water forinjection, and 1 mg/ml polysorbate 80. The formulation may contain20-110 mg, alternatively 20-90 mg of adalimumab or, alternatively, 30-80mg of the antibody. In one embodiment, the formulation contains 30 mg,31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg 69 mg, 70 mg, 71 mg,72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 101 mg, 102mg, 103 mg, 104 mg, 105 mg, 106 mg, 107 mg, 108 mg, 109 mg, or 110 mg ofthe antibody. Ranges including the aforementioned numbers are alsoincluded in the invention, e.g., 70-90 mg, 65-95 mg, or 60-85 mg.

Thus, the high antibody formulations and methods of the invention notonly overcome a number of known challenges for pharmaceuticalformulations, including high concentrations in a stable formulation, butalso possesses the added benefit of producing improved bioavailabilityor providing significantly low levels of pain when injected intopatients.

Another obstacle overcome by the formulations of the invention is theability to remain stable at room temperature (at about 25 degree C. orup to about 30 degrees C.). Such stability provides advantages for theuser of the antibody, providing for more flexible storage options, asthe constant need for refrigeration is unnecessary. Both the decreasedpain formulation and the increased bioavailability formulation(exemplified by formulations F3 and F4, respectively, in the Examplesbelow) are stable for at least 6 days at about 25 degrees C. or up toabout 30 degrees C. As described in more detail in the Examples, theformulations of the invention are stable at up to 30 degrees C. for atleast 6 days, at least 7 days, at least 8 days, at least 9 days, atleast 10 days, at least 11 days, at least 12 days, at least 13 days, andat least 14 days. Thus, the invention further provides formulationshaving extended (i.e., at least 6 days, 10 days or 14 days) shelf lifeat room temperature (or about 25 degrees C. or up to about 30 degreesC.). In one embodiment, the formulation of the invention is stable at 20to 32 degrees C. for at least 6 days. Temperatures intermediate to theabove recited concentrations are also intended to be part of thisinvention, i.e., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32degrees Celsius (C). Ranges including the aforementioned temperaturesare also included in the invention, e.g., 22-26 degrees C., 25-30degrees C., etc.

The formulations of the invention contain a high antibody concentration,including, for example, an antibody concentration of about 50 mg/mL, 55mg/mL, 60 mg/mL. 65 mg/mL, 70 mg/mL, 75 mg/ml, 80 mg/mL, 85 mg/mL, 90mg/mL, 95 mg/mL 100 mg/mL, 105 mg/mL, 110 mg/mL, 115 mg/mL (or higher)of a human anti-TNF-alpha antibody or antigen-binding fragment thereof.Accordingly, as described in the examples below, in one aspect of theinvention the liquid pharmaceutical formulations of the inventioncontain a human anti-TNF alpha antibody concentration of 50-100 mg/mL orgreater. In one embodiment, the formulations of the invention maycomprise an antibody concentration between about 1 mg/mL-150 mg/mL orabout 40 mg/mL-125 mg/mL. In one embodiment, the antibody concentrationof the formulation is 50-150 mg/ml, 55-150 mg/ml, 60-150 mg/ml, 65-150mg/ml, 70-150 mg/ml, 75-150 mg/ml, 80-150 mg/ml, 85-150 mg/ml, 90-150mg/ml, 90-110 mg/ml, 95-105 mg/ml, 95-150 mg/ml, 100-150 mg/ml, 105-150mg/ml, 110-150 mg/ml, 115-150 mg/ml, 120-150 mg/ml, 125-150 mg/ml,50-130 mg/ml, 75-125 mg/ml, etc. Concentrations and ranges intermediateto the above recited concentrations are also intended to be part of thisinvention (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150 mg/mL).

The formulations of the invention may contain an effective amount of theantibody. In one embodiment, an effective amount is about 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 mg ofthe human anti-TNFα antibody, or antigen-binding portion thereof. In oneembodiment, the formulations and methods of the invention comprise about20-100, about 20-90, about 30-90, about 30-100, about 60-100, about70-90, about 40-90, about 60-85 mg, or about 40-100 mg of a humananti-TNFα antibody, or antigen-binding portion thereof. In oneembodiment, the formulation contains 30 mg, 31 mg, 32 mg, 33 mg, 34 mg,35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65mg, 66 mg, 67 mg, 68 mg 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg,76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86mg, 87 mg, 88 mg, 89 mg, or 90 mg of the antibody. Ranges including theaforementioned numbers are also included in the invention, e.g., 70-90or 75-85 mg or 60-85 mg.

An important aspect of the formulations and methods of the invention isthe omission of a buffer and salt. Thus, in one embodiment, theformulations and methods of the invention do not contain any buffer(s)(e.g., citrate and phosphate) and salts. It should be noted, however,that although the preferred formulations of the invention do not containbuffers or salts (e.g., NaCl), a small amount of buffer and/or salt maybe present in the formulations. Thus, in one embodiment, theformulations of the invention do not contain detectable levels of abuffer(s) and/or a salt.

In one embodiment, the buffer(s) omitted from the formulations of theinvention (or those formulations for comparison which include abuffer(s)) may include citric acid (e.g., about 1.3-1.31 mg/mL or 1.305mg/mL). In another embodiment, the buffer system includes sodium citratedehydrate (e.g., about 0.27-0.33 mg/mL or about 0.305 mg/mL). In oneembodiment, the buffer system includes disodium phosphate dehydrate(e.g., about 1.5-1.56 mg/mL or about 1.53 mg/mL). In another embodiment,the buffer system includes sodium dihydrogen phosphate dihydrate (e.g.,about 0.83-0.89 mg/mL or about 0.86 mg/mL).

In one embodiment of the invention, the conductivity of the formulationmay be used to determine if a formulation has a buffer and/or salt. BothFormulation F3 and F4 (described in the working examples below) havebeen determined to have a conductivity of less than about 2 mS/cm, e.g.,about 0.70/cm. Thus, in one embodiment, the reduced pain and increasedbioavailability formulations of the invention have a conductivity ofless than about 2 mS/cm. In another embodiment, the formulations of theinvention have a conductivity of less than about 1 mS/cm.

In one embodiment, the formulation of the invention contains of a humananti-TNF alpha antibody, or antigen binding portion thereof, at aconcentration of about 100 mg/mL (or 75-125 mg/mL), a surfactant (e.g.,polysorbate 80), a polyol (e.g., sorbitol or mannitol), and has aconductivity of less than 2 mS/cm. In one embodiment, the formulation ofthe invention contains of a human anti-TNF alpha antibody, or antigenbinding portion thereof, at a concentration of about 100 mg/mL (or75-125 mg/mL), about 0.8-1.3 mg/ml of a surfactant (e.g., polysorbate80), less than about 50 mg/ml of a polyol (e.g., sorbitol or mannitol),and has a conductivity of less than 2 mS/cm.

In one embodiment, the formulation of the invention contains of a humananti-TNF alpha antibody, or antigen binding portion thereof, at aconcentration of about 100 mg/mL (or 75-125 mg/mL), a surfactant (e.g.,polysorbate 80), and has a conductivity of less than 2 mS/cm. In oneembodiment, the formulation of the invention contains of a humananti-TNF alpha antibody, or antigen binding portion thereof, at aconcentration of about 100 mg/mL (or 75-125 mg/mL), about 0.8-1.3 mg/mlof a surfactant (e.g., polysorbate 80), and has a conductivity of lessthan 2 mS/cm.

In another embodiment, the invention provides a stable formulationhaving a high concentration antibody, or antigen-binding portionthereof, wherein the antibody, or antigen has a hydrodynamic diameter(z-average) of less than about 4 nm or wherein the antibody, or antigenhas a hydrodynamic diameter (z-average) which is at least about 50% lessthan the hydrodynamic diameter of a buffered solution at the sameantibody concentration. In one embodiment, the antibody, or antigen hasa hydrodynamic diameter (z-average) of less than about 3 nm.

In one embodiment, the formulation of the invention contains of a humananti-TNF alpha antibody, or antigen binding portion thereof, at aconcentration of about 100 mg/mL (or 75-125 mg/mL), a surfactant (e.g.,polysorbate 80), a polyol (e.g., sorbitol or mannitol), and has ahydrodynamic diameter of less than 4 nm. In one embodiment, theformulation of the invention contains of a human anti-TNF alphaantibody, or antigen binding portion thereof, at a concentration ofabout 100 mg/mL (or 75-125 mg/mL), about 0.8-1.3 mg/ml of a surfactant(e.g., polysorbate 80), less than about 50 mg/ml of a polyol (e.g.,sorbitol or mannitol), and has a hydrodynamic diameter of less than 4nm.

In one embodiment, the formulation of the invention contains of a humananti-TNF alpha antibody, or antigen binding portion thereof, at aconcentration of about 100 mg/mL (or 75-125 mg/mL), a surfactant (e.g.,polysorbate 80), and has a hydrodynamic diameter of less than 4 nm. Inone embodiment, the formulation of the invention contains of a humananti-TNF alpha antibody, or antigen binding portion thereof, at aconcentration of about 100 mg/mL (or 75-125 mg/mL), about 0.8-1.3 mg/mlof a surfactant (e.g., polysorbate 80), and has a hydrodynamic diameterof less than 4 nm.

A detergent or surfactant is included in the antibody formulation of theinvention. Exemplary detergents include nonionic detergents such aspolysorbates (e.g. polysorbates 20, 80, etc.) or poloxamers (e.g.poloxamer 188). The amount of detergent added is such that it reducesaggregation of the formulated antibody and/or minimizes the formation ofparticulates in the formulation and/or reduces adsorption. In apreferred embodiment of the invention, the formulation includes asurfactant which is a polysorbate. In another preferred embodiment ofthe invention, the formulation contains the detergent polysorbate 80. Inone embodiment, the formulation contains between about 0.1 and about 2.0mg/mL of surfactant (e.g., polysorbate), e.g., about 1 mg/mL. Otherranges of polysorbate that may be included in the formulations of theinvention include 0.1 to 1.5 mg/ml, alternatively 0.2-1.4 mg/ml, 0.3-1.3mg/ml, 0.4-1.2 mg/ml, 0.5-1.1 mg/ml, 0.6-1.0 mg/ml, 0.6-1.1 mg/ml,0.7-1.1 mg/ml, 0.8-1.1 mg/ml, or 0.9-1.1 mg/ml. Values and rangesintermediate to the above recited concentrations are also intended to bepart of this invention, e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9. In addition, ranges ofvalues using a combination of any of the above-recited values as upperand/or lower limits are intended to be included, e.g., 0.3 to 1.1 mg/mL.

In one embodiment, the formulation of the invention consists essentiallyof a human anti-TNF alpha antibody, or antigen binding portion thereof,at a concentration of about 100 mg/mL (or 75-125 mg/mL), a surfactant(e.g., polysorbate 80), a polyol (e.g., sorbitol or mannitol), does notcontain a buffer(s) (e.g., citric acid monohydrate, sodium citrate,disodium phosphate dihydrate, and/or sodium dihydrogen phosphatedihydrate), and does not contain a salt (e.g., NaCl).

In certain embodiments, the otherwise identical formulation to which theformulation of the invention is compared for pain or bioavailabilitypurposes is a formulation containing adalimumab, sodium chloride,monobasic sodium phosphate dihydrate, dibasic sodium phosphatedihydrate, sodium citrate, citric acid monohydrate, mannitol,polysorbate 80, and Water for Injection.

The formulation herein may also be combined with one or more othertherapeutic agents as necessary for the particular indication beingtreated. In one embodiment, those with complementary activities that donot adversely affect the antibody of the formulation. Such therapeuticagents are suitably present in combination in amounts that are effectivefor the purpose intended. Additional therapeutic agents which can becombined with the formulation of the invention are further described inU.S. Pat. Nos. 6,090,382 and 6,258,562, each of which is incorporatedherein by reference. All formulations described herein may be used inthe methods of the invention as well.

III. Antibodies for Use in the Formulations and Methods of the Invention

The formulations and methods of the invention include an antibody, orantigen binding portion thereof, particularly an anti-TNFα antibody, orantigen binding portion or fragment thereof. Examples of antibodies thatmay be used in the invention include chimeric antibodies, non-humanantibodies, isolated human antibodies, humanized antibodies, and domainantibodies (dAbs). All antibodies described herein may be used in themethods of the invention as well.

In one embodiment, the formulations of the invention comprises anantibody, or antigen-binding portion thereof, which binds human TNFα,including, for example, adalimumab (also referred to as Humira,adalimumab, or D2E7; Abbott Laboratories). In a further embodiment, theformulation comprises an antibody that binds the same epitope asadalimumab, such as, but not limited to, an adalimumab biosimilarantibody. In one embodiment, the antibody is a human IgG1 antibodyhaving six CDRs corresponding to those of the light and heavy chain ofadalimumab.

In one embodiment, the invention features an isolated human antibody, orantigen-binding portion thereof, that binds to human TNF-alpha with highaffinity and a low off rate, and also has a high neutralizing capacity.In one embodiment, the human antibodies used in the invention arerecombinant, neutralizing human anti-hTNF-alpha antibodies.

In one aspect, the invention pertains to adalimumab antibodies andantibody portions, adalimumab-related antibodies and antibody portions,and other human antibodies and antibody portions with equivalentproperties to adalimumab, such as high affinity binding to hTNFa. withlow dissociation kinetics and high neutralizing capacity. In oneembodiment, the antibody, or antigen-binding fragment thereof, isdefined according to dissociation and binding characteristics similar toadalimumab. For example, the formulation may include a human antibodythat dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M or less, and ak_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determined by surfaceplasmon resonance. In another embodiment, the human antibody thatdissociates from human TNFα with a K_(d) of 1×10⁻⁹ M or less.

In one embodiment, the antibody, or antigen-binding fragment thereof, isa human antibody that dissociates from human TNFα with a K_(d) of 1×10⁻⁸M or less, and a k_(off) rate constant of 1×10⁻³ s⁻¹ or less, bothdetermined by surface plasmon resonance, and neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ Mor less. Examples and methods for making human, neutralizing antibodieswhich have a high affinity for human TNFα, including sequences of theantibodies, are described in U.S. Pat. No. 6,090,382 (referred to asD2E7), incorporated by reference herein. The amino sequences of D2E7 asdescribed in U.S. Pat. No. 6,090,382 are incorporated in their entiretyherein.

In one embodiment, the antibody used in the formulation of the inventionis D2E7, also referred to as HUMIRA™ or adalimumab (the amino acidsequence of the D2E7 VL region is shown in SEQ ID NO: 1; the amino acidsequence of the D2E7 VH region is shown in SEQ ID NO: 2). The propertiesof D2E7 (adalimumab/HUMIRA®) have been described in Salfeld et al., U.S.Pat. Nos. 6,090,382, 6,258,562, and 6,509,015, which are eachincorporated by reference herein.

In one embodiment, the human TNF-alpha, or an antigen-binding portionthereof, dissociates from human TNF-alpha with a K_(d) of 1×10⁻⁸ M orless and a k_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determinedby surface plasmon resonance, and neutralizes human TNF-alphacytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ Mor less. In one embodiment, the isolated human antibody, orantigen-binding portion thereof, dissociates from human TNF-alpha with ak_(off) of 5×10⁻⁴ s⁻¹ or less; or, in one embodiment, with a k_(off) of1×10⁻⁴ s⁻¹ or less. In one embodiment, the isolated human antibody, orantigen-binding portion thereof, neutralizes human TNF-alphacytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ Mor less; or, in one embodiment, with an IC₅₀ of 1×10⁻⁹ M or less; or, inone embodiment, with an IC₅₀ of 1×10⁻¹⁰ M or less. In one embodiment,the antibody is an isolated human recombinant antibody, or anantigen-binding portion thereof.

It is well known in the art that antibody heavy and light chain CDR3domains play an important role in the binding specificity/affinity of anantibody for an antigen. Accordingly, in another aspect, the antibodyused in the formulation of the invention has slow dissociation kineticsfor association with hTNF-alpha and has light and heavy chain CDR3domains that structurally are identical to or related to those ofadalimumab. Position 9 of the adalimumab VL CDR3 can be occupied by Alaor Thr without substantially affecting the Koff. Accordingly, aconsensus motif for the adalimumab VL CDR3 comprises the amino acidsequence: Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID NO: 3). Additionally, position12 of the adalimumab VH CDR3 can be occupied by Tyr or Asn, withoutsubstantially affecting the k_(off). Accordingly, a consensus motif forthe adalimumab VH CDR3 comprises the amino acid sequence:V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID NO: 4). Moreover, as demonstrated inExample 2 of U.S. Pat. No. 6,090,382, the CDR3 domain of the adalimumabheavy and light chains is amenable to substitution with a single alanineresidue (at position 1, 4, 5, 7 or 8 within the VL CDR3 or at position2, 3, 4, 5, 6, 8, 9, 10 or 11 within the VH CDR3) without substantiallyaffecting the k_(off). Still further, the skilled artisan willappreciate that, given the amenability of the adalimumab VL and VH CDR3domains to substitutions by alanine, substitution of other amino acidswithin the CDR3 domains may be possible while still retaining the lowoff rate constant of the antibody, in particular substitutions withconservative amino acids. In one embodiment, no more than one to fiveconservative amino acid substitutions are made within the adalimumab VLand/or VH CDR3 domains. In one embodiment, no more than one to threeconservative amino acid substitutions are made within the adalimumab VLand/or VH CDR3 domains. Additionally, conservative amino acidsubstitutions should not be made at amino acid positions critical forbinding to hTNF alpha. Positions 2 and 5 of the adalimumab VL CDR3 andpositions 1 and 7 of the adalimumab VH CDR3 appear to be critical forinteraction with hTNF alpha, and thus, conservative amino acidsubstitutions preferably are not made at these positions (although analanine substitution at position 5 of the adalimumab VL CDR3 isacceptable, as described above) (see U.S. Pat. No. 6,090,382).

Accordingly, in one embodiment, the antibody or antigen-binding portionthereof, used in the formulation of the invention contains the followingcharacteristics:

a) dissociates from human TNFα with a k_(off) rate constant of 1×10⁻³s⁻¹ or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

In certain embodiments, the antibody or antigen-binding portion thereof,dissociates from human TNF-alpha with a k_(off) of 5×10⁻⁴ s⁻¹ or less.In certain embodiments, the antibody or antigen-binding portion thereof,dissociates from human TNF-alpha with a k_(off) of 1×10⁻⁴ s⁻¹ or less.

In yet another embodiment, the antibody or antigen-binding portionthereof contains a light chain variable region (LCVR) having a CDR3domain comprising the amino acid sequence of SEQ ID NO: 3, or modifiedfrom SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5,7 or 8, and with a heavy chain variable region (HCVR) having a CDR3domain comprising the amino acid sequence of SEQ ID NO: 4, or modifiedfrom SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4,5, 6, 8, 9, 10 or 11. In one embodiment, the LCVR further has a CDR2domain comprising the amino acid sequence of SEQ ID NO: 5 (i.e., theD2E7 VL CDR2) and the HCVR further has a CDR2 domain comprising theamino acid sequence of SEQ ID NO: 6 (i.e., the D2E7 VH CDR2). In oneembodiment, the LCVR further has CDR1 domain comprising the amino acidsequence of SEQ ID NO: 7 (i.e., the D2E7 VL CDR1) and the HCVR has aCDR1 domain comprising the amino acid sequence of SEQ ID NO: 8 (i.e.,the D2E7 VH CDR1). The framework regions for VL may be from the VκIhuman germline family, or from the A20 human germline Vk gene, or fromthe adalimumab VL framework sequences shown in FIGS. 1A and 1B of U.S.Pat. No. 6,090,382. The framework regions for VH may be from the VH3human germline family, or from the DP-31 human germline VH gene, or fromthe D2E7 VH framework sequences shown in FIGS. 2A and 2B of U.S. Pat.No. 6,090,382. Nucleic acid sequences corresponding to the adalimumablight and heavy variable regions are described in SEQ ID NOs: 36 and 37,respectively.

Accordingly, in another embodiment, the antibody or antigen-bindingportion thereof contains a light chain variable region (LCVR) comprisingthe amino acid sequence of SEQ ID NO: 1 (i.e., the adalimumab VL) and aheavy chain variable region (HCVR) comprising the amino acid sequence ofSEQ ID NO: 2 (i.e., the adalimumab VH). In certain embodiments, theantibody comprises a heavy chain constant region, such as an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region. In certainembodiments, the heavy chain constant region is an IgG1 heavy chainconstant region or an IgG4 heavy chain constant region. Furthermore, theantibody can comprise a light chain constant region, either a kappalight chain constant region or a lambda light chain constant region. Inone embodiment, the antibody comprises a kappa light chain constantregion. Alternatively, the antibody portion can be, for example, a Fabfragment or a single chain Fv fragment.

In still other embodiments, the invention includes uses of an isolatedhuman antibody, or an antigen-binding portion thereof, containingadalimumab-related VL and VH CDR3 domains. For example, antibodies orantigen-binding portions thereof may have a light chain variable region(LCVR) having a CDR3 domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 orwith a heavy chain variable region (HCVR) having a CDR3 domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQID NO: 35.

In one embodiment, the TNFα antibody used in the invention includes thechimeric antibody infliximab (Remicade®, Johnson and Johnson; describedin U.S. Pat. No. 5,656,272, incorporated by reference herein), CDP571 (ahumanized monoclonal anti-TNF-alpha IgG4 antibody), CDP 870 (a humanizedmonoclonal anti-TNF-alpha antibody fragment), an anti-TNF dAb (Peptech),or CNTO 148 (golimumab; Medarex and Centocor, see WO 02/12502).Additional TNF antibodies which may be used in the invention aredescribed in U.S. Pat. Nos. 6,593,458; 6,498,237; 6,451,983; and6,448,380, each of which is incorporated by reference herein.

An antibody, or antibody portion, used in the methods and compositionsof the invention, can be prepared by recombinant expression ofimmunoglobulin light and heavy chain genes in a host cell. To express anantibody recombinantly, a host cell is transfected with one or morerecombinant expression vectors carrying DNA fragments encoding theimmunoglobulin light and heavy chains of the antibody such that thelight and heavy chains are expressed in the host cell and, preferably,secreted into the medium in which the host cells are cultured, fromwhich medium the antibodies can be recovered. Standard recombinant DNAmethodologies are used to obtain antibody heavy and light chain genes,incorporate these genes into recombinant expression vectors andintroduce the vectors into host cells, such as those described inSambrook, Fritsch and Maniatis (eds), Molecular Cloning; A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M.et al. (eds.) Current Protocols in Molecular Biology, Greene PublishingAssociates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al.

To express an anti-TNFa antibody, e.g., adalimumab (D2E7) or anadalimumab (D2E7)-related antibody, DNA fragments encoding the light andheavy chain variable regions are first obtained. These DNAs can beobtained by amplification and modification of germline light and heavychain variable sequences using the polymerase chain reaction (PCR).Germline DNA sequences for human heavy and light chain variable regiongenes are known in the art (see e.g., the “Vbase” human germlinesequence database; see also Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson, I.M., et al. (1992) “The Repertoire of Human Germline VH Sequences Revealsabout Fifty Groups of VH Segments with Different Hypervariable Loops” J.Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory ofHuman Germ-line V78 Segments Reveals a Strong Bias in their Usage” Eur.J. Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference). For example, to obtain a DNA fragmentencoding the heavy chain variable region of D2E7, or a D2E7-relatedantibody, a member of the VH3 family of human germline VH genes isamplified by standard PCR. In certain embodiments, the DP-31 VH germlinesequence is amplified. To obtain a DNA fragment encoding the light chainvariable region of D2E7, or a D2E7-related antibody, a member of the VκIfamily of human germline VL genes is amplified by standard PCR. Incertain embodiments, the A20 VL germline sequence is amplified. PCRprimers suitable for use in amplifying the DP-31 germline VH and A20germline VL sequences can be designed based on the nucleotide sequencesdisclosed in the references cited supra, using standard methods.

Once the germline VH and VL fragments are obtained, these sequences canbe mutated to encode the anti-TNFa antibody amino acid sequencesdisclosed herein. The amino acid sequences encoded by the germline VHand VL DNA sequences are first compared to the anti-TNFa antibody VH andVL amino acid sequences to identify amino acid residues in the anti-TNFaantibody sequence that differ from germline. Then, the appropriatenucleotides of the germline DNA sequences are mutated such that themutated germline sequence encodes the anti-TNFa antibody amino acidsequence, using the genetic code to determine which nucleotide changesshould be made. Mutagenesis of the germline sequences is carried out bystandard methods, such as PCR-mediated mutagenesis (in which the mutatednucleotides are incorporated into the PCR primers such that the PCRproduct contains the mutations) or site-directed mutagenesis.

Moreover, it should be noted that if the “germline” sequences obtainedby PCR amplification encode amino acid differences in the frameworkregions from the true germline configuration (i.e., differences in theamplified sequence as compared to the true germline sequence, forexample as a result of somatic mutation), it may be desirable to changethese amino acid differences back to the true germline sequences (i.e.,“backmutation” of framework residues to the germline configuration).

Once DNA fragments encoding the anti-TNFα antibody VH and VL segmentsare obtained (e.g., by amplification and mutagenesis of germline VH andVL genes, as described above), these DNA fragments can be furthermanipulated by standard recombinant DNA techniques, for example toconvert the variable region genes to full-length antibody chain genes,to Fab fragment genes or to a scFv gene. In these manipulations, a VL-or VH-encoding DNA fragment is operatively linked to another DNAfragment encoding another protein, such as an antibody constant regionor a flexible linker. The term “operatively linked,” as used in thiscontext, is intended to mean that the two DNA fragments are joined suchthat the amino acid sequences encoded by the two DNA fragments remainin-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region. In one embodiment, the lightchain constant region is a kappa constant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

To express the antibodies, or antibody portions used in the invention,DNAs encoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or, more typically, both genesare inserted into the same expression vector. The antibody genes areinserted into the expression vector by standard methods (e.g., ligationof complementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present).Prior to insertion of the anti-TNFa antibody light or heavy chainsequences, the expression vector may already carry antibody constantregion sequences. For example, one approach to converting the anti-TNFaantibody VH and VL sequences to full-length antibody genes is to insertthem into expression vectors already encoding heavy chain constant andlight chain constant regions, respectively, such that the VH segment isoperatively linked to the CH segment(s) within the vector and the VLsegment is operatively linked to the CL segment within the vector.Additionally or alternatively, the recombinant expression vector canencode a signal peptide that facilitates secretion of the antibody chainfrom a host cell. The antibody chain gene can be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino terminus ofthe antibody chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors used in the invention may carryadditional sequences, such as sequences that regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. The selectable marker gene facilitates selection of hostcells into which the vector has been introduced (see e.g., U.S. Pat.Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Preferred selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr-hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ispreferably in eukaryotic cells. In one embodiment, mammalian host cells,is the most preferred because such eukaryotic cells, and in particularmammalian cells, are more likely than prokaryotic cells to assemble andsecrete a properly folded and immunologically active antibody.Prokaryotic expression of antibody genes has been reported to beineffective for production of high yields of active antibody (Boss, M.A. and Wood, C. R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more, in one embodiment, secretion ofthe antibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It is understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain (but notboth) of an antibody of this invention. Recombinant DNA technology mayalso be used to remove some or all of the DNA encoding either or both ofthe light and heavy chains that is not necessary for binding to hTNFalpha. The molecules expressed from such truncated DNA molecules arealso encompassed by the antibodies of the invention. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the invention and the other heavy and lightchain are specific for an antigen other than hTNF alpha by crosslinkingan antibody of the invention to a second antibody by standard chemicalcrosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells areculture to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.

In view of the foregoing, nucleic acid, vector and host cellcompositions that can be used for recombinant expression of theantibodies and antibody portions used in the invention include nucleicacids, and vectors comprising said nucleic acids, comprising the humanTNF alpha antibody adalimumab (D2E7). The nucleotide sequence encodingthe D2E7 light chain variable region is shown in SEQ ID NO: 36. The CDR1domain of the LCVR encompasses nucleotides 70-102, the CDR2 domainencompasses nucleotides 148-168 and the CDR3 domain encompassesnucleotides 265-291. The nucleotide sequence encoding the D2E7 heavychain variable region is shown in SEQ ID NO: 37. The CDR1 domain of theHCVR encompasses nucleotides 91-105, the CDR2 domain encompassesnucleotides 148-198 and the CDR3 domain encompasses nucleotides 295-330.It will be appreciated by the skilled artisan that nucleotide sequencesencoding D2E7-related antibodies, or portions thereof (e.g., a CDRdomain, such as a CDR3 domain), can be derived from the nucleotidesequences encoding the D2E7 LCVR and HCVR using the genetic code andstandard molecular biology techniques.

In one embodiment, the liquid pharmaceutical formulation comprises ahuman TNF alpha antibody, or antigen-binding portion thereof, that is abioequivalent or biosimilar to the antibody adalimumab. In oneembodiment, a biosimilar antibody is an antibody which shows noclinically meaningful difference when compared to a reference antibody,e.g., adalimumab. A biosimilar antibody has equivalent safety, purity,and potency as a reference antibody, e.g., adalimumab.

IV. Administration of the Formulations of the Invention for Treatment ofTNFa-Related Disorders

An advantage of the formulations of the invention is that they may beused to deliver a high concentration of an anti-TNF alpha antibody, orantigen-binding portion, (e.g., adalimumab) to a subject subcutaneouslysuch that either pain upon injection is decreased or the bioavailabilityof the antibody is improved. Thus, in one embodiment, the formulation ofthe invention is delivered to a subject subcutaneously. In oneembodiment, the subject administers the formulation to himself/herself(self-administration).

In one embodiment, an effective amount of the formulation isadministered. An example of an effective amount of the formulation is anamount sufficient to inhibit detrimental TNF-alpha activity or treat adisorder in which TNF alpha activity is detrimental.

As used herein, the term “a disorder in which TNF-alpha activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of TNF-alpha. in a subject suffering from thedisorder has been shown to be or is suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder. Accordingly, a disorder inwhich TNF-alpha. activity is detrimental is a disorder in whichinhibition of TNF-alpha activity is expected to alleviate the symptomsand/or progression of the disorder. Such disorders may be evidenced, forexample, by an increase in the concentration of TNF-alpha. in abiological fluid of a subject suffering from the disorder (e.g., anincrease in the concentration of TNF-alpha. in serum, plasma, synovialfluid, etc. of the subject), which can be detected, for example, usingan anti-TNF-alpha. antibody.

In one embodiment, the effective amount of antibody may be determinedaccording to a strictly weight based dosing scheme (e.g., mg/kg) or maybe a total body dose (also referred to as a fixed dose) which isindependent of weight. In one embodiment, an effective amount of theantibody is about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or about 100 mg of the human anti-TNFα antibody, orantigen-binding portion thereof. In one embodiment, an effective amountof the antibody is about 20-100, about 20-90, about 30-90, about 30-100,about 60-100, about 70-90, about 40-90, about 60-85 mg, or about 40-100mg. In one embodiment, the formulation contains an effective amount ofthe antibody of 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg,38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99mg, or 100 mg of the antibody. Ranges including the aforementionednumbers are also included in the invention, e.g., 70-90 or 75-85 mg or60-85 mg.

In one example, an effective amount of the formulation is 0.4 mL or 0.8mL of the formulation containing a total body dose of about 80 mg ofantibody (i.e., 0.8 mL of a 100 mg/mL antibody formulation of theinvention). In another example, an effective amount of the formulationis 0.4 mL of the formulation of the invention containing a total bodydose of about 40 mg of antibody (i.e., 0.4 mL of a 100 mg/mL antibodyformulation of the invention). In yet another example, an effectiveamount of the formulation is twice 0.8 mL of the formulation containinga total body dose of about 160 mg of antibody (i.e., two unitscontaining 0.8 mL each of a 100 mg/mL antibody formulation of theinvention). In a further example, an effective amount of the formulationis 0.2 mL of the formulation of the invention containing a total bodydose of about 20 mg of antibody (i.e., 0.2 mL of a 100 mg/mL antibodyformulation of the invention). Alternatively, an effective amount may bedetermined according to a weight-based fixed dosing regimen (see, e.g.,WO 2008/154543, incorporated by reference herein).

In one embodiment, the TNF-alpha is human TNF-alpha and the subject is ahuman subject. Alternatively, the subject can be a mammal expressing aTNF-alpha with which an antibody of the invention cross-reacts. Stillfurther the subject can be a mammal into which has been introducedhTNF-alpha (e.g., by administration of hTNF-alpha or by expression of anhTNF-alpha transgene).

A formulation of the invention may be administered to a human subjectfor therapeutic purposes (discussed further below). In one embodiment ofthe invention, the liquid pharmaceutical formulation is easilyadministratable, which includes, for example, a formulation which isself-administered by the patient. In one embodiment, the formulation ofthe invention is administered through subcutaneous injection, such assingle use subcutaneous injection. Moreover, a formulation of theinvention can be administered to a non-human mammal expressing aTNF-alpha with which the antibody cross-reacts (e.g., a primate, pig ormouse) for veterinary purposes or as an animal model of human disease.Regarding the latter, such animal models may be useful for evaluatingthe therapeutic efficacy of antibodies of the invention (e.g., testingof dosages and time courses of administration).

The formulations of the invention may be administered according to acertain dosing schedule. For example, the formulations may beadministered according to a weekly, biweekly, or monthly dosing regimen.Alternatively, the formulation may be administered once every threeweeks. In one embodiment, the formulations and methods compriseadministration to the subject of a human anti-TNFα antibody according toa periodicity selected from the group consisting of weekly, biweekly,every three weeks, and monthly.

In one embodiment, the liquid aqueous formulation of the invention maybe administered to a subject via, for example, a prefilled syringe, anautoinjector pen, or a needle-free administration device. Thus, theinvention also features an autoinjector pen, a prefilled syringe, or aneedle-free administration device comprising the liquid aqueousformulation of the invention. In one embodiment, the invention featuresa delivery device comprising a dose of the formulation comprising 100mg/mL a human TNF alpha antibody, or antigen-binding portion thereof,e.g., an autoinjector pen or prefilled syringe comprises a dose of about19 mg, 20, mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg,29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99mg, 100 mg, 101 mg, 102 mg, 103 mg, 104 mg, 105 mg, etc. of theformulation. In one embodiment, the syringe or autoinjector contains60-100 mg, 70-90 mg, or about 80 mg of the antibody.

In one embodiment, the formulations of the invention may be selfadministered using, e.g., a preloaded syringe or an automatic injectiondevice. Automatic injection devices offer an alternative tomanually-operated syringes for delivering therapeutic agents intopatients' bodies and allowing patients to self-administer injections.Automatic injection devices are described, for example, in the followingpublications, each of which is hereby incorporated herein by referenceWO 2008/005315, WO 2010/127146, WO 2006/000785, WO 2011/075524, WO2005/113039, WO 2011/075524.

Accordingly, in one embodiment, the present invention providespre-filled syringes or autoinjector devices containing the formulationsof the invention, as well as use of pre-filled syringes or autoinjectordevices comprising the formulations described herein in the methods ofthe invention.

In one embodiment, the formulation of the invention is used to treatdisorders in which TNF alpha activity is detrimental. As used herein,the term “a disorder in which TNF-alpha activity is detrimental” isintended to include diseases and other disorders in which the presenceof TNF-alpha in a subject suffering from the disorder has been shown tobe or is suspected of being either responsible for the pathophysiologyof the disorder or a factor that contributes to a worsening of thedisorder. Accordingly, a disorder in which TNF-alpha activity isdetrimental is a disorder in which inhibition of TNF-alpha activity isexpected to alleviate the symptoms and/or progression of the disorder.Such disorders may be evidenced, for example, by an increase in theconcentration of TNF-alpha in a biological fluid of a subject sufferingfrom the disorder (e.g., an increase in the concentration of TNF-alphain serum, plasma, synovial fluid, etc. of the subject), which can bedetected, for example, using an anti-TNF-alpha antibody as describedabove.

There are numerous examples of disorders in which TNF-alpha activity isdetrimental. Examples in which TNF-alpha activity is detrimental arealso described in U.S. Pat. Nos. 6,015,557; 6,177,077; 6,379,666;6,419,934; 6,419,944; 6,423,321; 6,428,787; and 6,537,549; and PCTPublication Nos. WO 00/50079 and WO 01/49321, the entire contents of allof which are incorporated herein by reference. The formulations of theinvention may also be used to treat disorders in which TNF alphaactivity is detrimental as described in U.S. Pat. Nos. 6,090,382,6,258,562 and U.S. Patent Application Publication No. US20040126372, theentire contents of all of which are incorporated herein by reference.

The use of the formulations of the invention in the treatment ofspecific exemplary disorders is discussed further below:

A. Sepsis

The formulations and methods of the invention may be used to treatsubjects having sepsis. Tumor necrosis factor has an established role inthe pathophysiology of sepsis, with biological effects that includehypotension, myocardial suppression, vascular leakage syndrome, organnecrosis, stimulation of the release of toxic secondary mediators andactivation of the clotting cascade (see e.g., Tracey, K. J. and Cerami,A. (1994) Annu. Rev. Med. 45:491-503; Russell, D and Thompson, R. C.(1993) Curr. Opin. Biotech. 4:714-721). Accordingly, the formulation ofthe invention can be used to treat sepsis in any of its clinicalsettings, including septic shock, endotoxic shock, gram negative sepsisand toxic shock syndrome.

Furthermore, to treat sepsis, the formulation of the invention can becoadministered with one or more additional therapeutic agents that mayfurther alleviate sepsis, such as an interleukin-1 inhibitor (such asthose described in PCT Publication Nos. WO 92/16221 and WO 92/17583),the cytokine interleukin-6 (see e.g., PCT Publication No. WO 93/11793)or an antagonist of platelet activating factor (see e.g., EuropeanPatent Application Publication No. EP 374 510).

Additionally, in one embodiment, the formulation of the invention isadministered to a human subject within a subgroup of sepsis patientshaving a serum or plasma concentration of IL-6 above 500 pg/ml; or, inone embodiment, 1000 pg/ml, at the time of treatment (see PCTPublication No. WO 95/20978).

B. Autoimmune Diseases

The formulations and methods of the invention may be used to treatsubjects having an autoimmune disease. Tumor necrosis factor has beenimplicated in playing a role in the pathophysiology of a variety ofautoimmune diseases. For example, TNF-alpha has been implicated inactivating tissue inflammation and causing joint destruction inrheumatoid arthritis (see e.g., Tracey and Cerami, supra; Arend, W. P.and Dayer, J-M. (1995) Arth. Rheum. 38:151-160; Fava, R. A., et al.(1993) Clin. Exp. Immunol. 94:261-266). TNF-alpha also has beenimplicated in promoting the death of islet cells and in mediatinginsulin resistance in diabetes (see e.g., Tracey and Cerami, supra; PCTPublication No. WO 94/08609). TNF-alpha also has been implicated inmediating cytotoxicity to oligodendrocytes and induction of inflammatoryplaques in multiple sclerosis (see e.g., Tracey and Cerami, supra). Alsoincluded in autoimmune diseases that may be treated using theformulations and methods of the invention is juvenile idiopathicarthritis (JIA) (also referred to as juvenile rheumatoid arthritis) (seeGrom et al. (1996) Arthritis Rheum. 39:1703; Mangge et al. (1995)Arthritis Rheum. 8:211).

The formulation of the invention can be used to treat autoimmunediseases, in particular those associated with inflammation, includingrheumatoid arthritis, rheumatoid spondylitis (also referred to asankylosing spondylitis), osteoarthritis and gouty arthritis, allergy,multiple sclerosis, autoimmune diabetes, autoimmune uveitis, juvenileidiopathic arthritis (also referred to as juvenile rheumatoidarthritis), and nephrotic syndrome.

C. Infectious Diseases

The formulations and methods of the invention may be used to treatsubjects having an infectious disease. Tumor necrosis factor has beenimplicated in mediating biological effects observed in a variety ofinfectious diseases. For example, TNF-alpha has been implicated inmediating brain inflammation and capillary thrombosis and infarction inmalaria (see e.g., Tracey and Cerami, supra). TNF-alpha also has beenimplicated in mediating brain inflammation, inducing breakdown of theblood-brain barrier, triggering septic shock syndrome and activatingvenous infarction in meningitis (see e.g., Tracey and Cerami, supra).TNF-alpha also has been implicated in inducing cachexia, stimulatingviral proliferation and mediating central nervous system injury inacquired immune deficiency syndrome (AIDS) (see e.g., Tracey and Cerami,supra). Accordingly, the antibodies, and antibody portions, of theinvention, can be used in the treatment of infectious diseases,including bacterial meningitis (see e.g., European Patent ApplicationPublication No. EP 585 705), cerebral malaria, AIDS and AIDS-relatedcomplex (ARC) (see e.g., European Patent Application Publication No. EP230 574), as well as cytomegalovirus infection secondary totransplantation (see e.g., Fietze, E., et al. (1994) Transplantation58:675-680). The formulation of the invention, also can be used toalleviate symptoms associated with infectious diseases, including feverand myalgias due to infection (such as influenza) and cachexia secondaryto infection (e.g., secondary to AIDS or ARC).

D. Transplantation

The formulations and methods of the invention may be used to treatsubjects having a transplantation. Tumor necrosis factor has beenimplicated as a key mediator of allograft rejection and graft versushost disease (GVHD) and in mediating an adverse reaction that has beenobserved when the rat antibody OKT3, directed against the T cellreceptor CD3 complex, is used to inhibit rejection of renal transplants(see e.g., Tracey and Cerami, supra; Eason, J. D., et al. (1995)Transplantation 59:300-305; Suthanthiran, M. and Strom, T. B. (1994) NewEngl. J. Med. 331:365-375). Accordingly, the formulations of theinvention can be used to inhibit transplant rejection, includingrejections of allografts and xenografts and to inhibit GVHD. Althoughthe antibody or antibody portion may be used alone, it can be used incombination with one or more other agents that inhibit the immuneresponse against the allograft or inhibit GVHD. For example, in oneembodiment, the formulations of the invention are used in combinationwith OKT3 to inhibit OKT3-induced reactions. In another embodiment, theformulation of the invention is used in combination with one or moreantibodies directed at other targets involved in regulating immuneresponses, such as the cell surface molecules CD25 (interleukin-2receptor-.alpha.), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4,CD80 (B7-1) and/or CD86 (B7-2). In yet another embodiment, theformulation of the invention is used in combination with one or moregeneral immunosuppressive agents, such as cyclosporin A or FK506.

E. Malignancy

The formulations and methods of the invention may be used to treatsubjects having cancer or a malignant tumor. Tumor necrosis factor hasbeen implicated in inducing cachexia, stimulating tumor growth,enhancing metastatic potential and mediating cytotoxicity inmalignancies (see e.g., Tracey and Cerami, supra). Accordingly, theformulations of the invention can be used in the treatment ofmalignancies, to inhibit tumor growth or metastasis and/or to alleviatecachexia secondary to malignancy. The formulation of the invention maybe administered systemically or locally to the tumor site.

F. Pulmonary Disorders

The formulations and methods of the invention may be used to treatsubjects having a pulmonary disease. Tumor necrosis factor has beenimplicated in the pathophysiology of adult respiratory distresssyndrome, including stimulating leukocyte-endothelial activation,directing cytotoxicity to pneumocytes and inducing vascular leakagesyndrome (see e.g., Tracey and Cerami, supra). Accordingly, theformulations of the invention can be used to treat various pulmonarydisorders, including adult respiratory distress syndrome (see e.g., PCTPublication No. WO 91/04054), shock lung, chronic pulmonary inflammatorydisease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis. Theformulation of the invention may be administered systemically or locallyto the lung surface, for example as an aerosol.

G. Intestinal Disorders

The formulations and methods of the invention may be used to treatsubjects having an intestinal disorder. Tumor necrosis factor has beenimplicated in the pathophysiology of inflammatory bowel disorders (seee.g., Tracy, K. J., et al. (1986) Science 234:470-474; Sun, X-M., et al.(1988) J. Clin. Invest. 81:1328-1331; MacDonald, T. T., et al. (1990)Clin. Exp. Immunol. 81:301-305) Chimeric murine anti-hTNF-alphaantibodies have undergone clinical testing for treatment of Crohn'sdisease (van Dullemen, H. M., et al. (1995) Gastroenterology109:129-135). The formulation of the invention, also can be used totreat intestinal disorders, such as idiopathic inflammatory boweldisease, which includes two syndromes, Crohn's disease and ulcerativecolitis. In one embodiment, the formulation of the invention is used totreat Crohn's disease. In one embodiment, the formulation of theinvention is used to treat ulcerative colitis.

H. Cardiac Disorders

The formulations and methods of the invention, also can be used to treatvarious cardiac disorders, including ischemia of the heart (see e.g.,European Patent Application Publication No. EP 453 898) and heartinsufficiency (weakness of the heart muscle)(see e.g., PCT PublicationNo. WO 94/20139).

I. Spondyloarthropathies

The formulations and methods of the invention may also be used to treatsubjects who have a spondyloarthropathy, including, for example, anaxial spondyloarthropathy. TNFα has been implicated in thepathophysiology of a wide variety of disorders, including inflammatorydiseases such as spondyloarthopathies (see e.g., Moeller, A., et al.(1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.;European Patent Publication No. 260 610 B1 by Moeller, A). In oneembodiment, the spondyloarthropathy is an axial spondyloarthropathy.Other examples of spondyloarthropathies which can be treated with theTNFα antibody of the invention are described below:

1. Psoriatic Arthritis

The formulations and methods of the invention may also be used to treatsubjects who have psoriatic arthritis. Tumor necrosis factor has beenimplicated in the pathophysiology of psoriatic arthritis (Partsch et al.(1998) Ann Rheum Dis. 57:691; Ritchlin et al. (1998) J Rheumatol.25:1544). As referred to herein, psoriatic arthritis (PsA) or psoriasisassociated with the skin, refers to chronic inflammatory arthritis whichis associated with psoriasis. Psoriasis is a common chronic skincondition that causes red patches on the body. About 1 in 20 individualswith psoriasis will develop arthritis along with the skin condition, andin about 75% of cases, psoriasis precedes the arthritis. PsA exhibitsitself in a variety of ways, ranging from mild to severe arthritis,wherein the arthritis usually affects the fingers and the spine. Whenthe spine is affected, the symptoms are similar to those of ankylosingspondylitis, as described above.

PsA is sometimes associated with arthritis mutilans. Arthritis mutilansrefers to a disorder which is characterized by excessive bone erosionresulting in a gross, erosive deformity which mutilates the joint. Inone embodiment, formulations and methods of the invention can be used totreat arthritis mutilans.

2. Reactive Arthritis/Reiter's Syndrome

The formulations and methods of the invention may also be used to treatsubjects who have Reiter's syndrome or reactive arthritis. Tumornecrosis factor has been implicated in the pathophysiology of reactivearthritis, which is also referred to as Reiter's syndrome (Braun et al.(1999) Arthritis Rheum. 42(10):2039). Reactive arthritis (ReA) refers toarthritis which complicates an infection elsewhere in the body, oftenfollowing enteric or urogenital infections. ReA is often characterizedby certain clinical symptoms, including inflammation of the joints(arthritis), urethritis, conjunctivitis, and lesions of the skin andmucous membranes. In addition, ReA can occurs following infection with asexually transmitted disease or dysenteric infection, includingchlamydia, campylobacter, salmonella, or yersinia.

3. Undifferentiated Spondyloarthropathies

The formulations and methods of the invention may also be used to treatsubjects who have an undifferentiated spondyloarthropathy (see Zeidleret al. (1992) Rheum Dis Clin North Am. 18:187). Other terms used todescribe undifferentiated spondyloarthropathies include seronegativeoligoarthritis and undifferentiated oligoarthritis. Undifferentiatedspondyloarthropathies, as used herein, refers to a disorder wherein thesubject demonstrates only some of the symptoms associated with aspondyloarthropathy. This condition is usually observed in young adultswho do not have IBD, psoriasis, or the classic symptoms of AS orReiter's syndrome. In some instances, undifferentiatedspondyloarthropathies may be an early indication of AS.

J. Skin and Nail Disorders

In one embodiment, the formulations and methods of the invention areused to treat a skin and/or a nail disorder. As used herein, the term“skin and nail disorder in which TNFα activity is detrimental” isintended to include skin and/or nail disorders and other disorders inwhich the presence of TNF alpha in a subject suffering from the disorderhas been shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder, e.g., psoriasis. An example of a skindisorder which may be treated using the formulation of the invention ispsoriasis. In one embodiment, the formulation of the invention is usedto treat plaque psoriasis. Tumor necrosis factor has been implicated inthe pathophysiology of psoriasis (Takematsu et al. (1989) Arch DermatolRes. 281:398; Victor and Gottlieb (2002) J Drugs Dermatol. 1(3):264).

1. Psoriasis

The formulations and methods of the invention may be used to treatsubjects having psoriasis, including subjects having plaque psoriasis.Tumor necrosis factor has been implicated in the pathophysiology ofpsoriasis (Takematsu et al. (1989) Arch Dermatol Res. 281:398; Victorand Gottlieb (2002) J Drugs Dermatol. 1(3):264). Psoriasis is describedas a skin inflammation (irritation and redness) characterized byfrequent episodes of redness, itching, and thick, dry, silvery scales onthe skin. In particular, lesions are formed which involve primary andsecondary alterations in epidermal proliferation, inflammatory responsesof the skin, and an expression of regulatory molecules such aslymphokines and inflammatory factors. Psoriatic skin is morphologicallycharacterized by an increased turnover of epidermal cells, thickenedepidermis, abnormal keratinization, inflammatory cell infiltrates intothe epidermis and polymorphonuclear leukocyte and lymphocyteinfiltration into the epidermis layer resulting in an increase in thebasal cell cycle. Psoriasis often involves the nails, which frequentlyexhibit pitting, separation of the nail, thickening, and discoloration.Psoriasis is often associated with other inflammatory disorders, forexample arthritis, including rheumatoid arthritis, inflammatory boweldisease (IBD), and Crohn's disease.

Evidence of psoriasis is most commonly seen on the trunk, elbows, knees,scalp, skin folds, or fingernails, but it may affect any or all parts ofthe skin. Normally, it takes about a month for new skin cells to move upfrom the lower layers to the surface. In psoriasis, this process takesonly a few days, resulting in a build-up of dead skin cells andformation of thick scales. Symptoms of psoriasis include: skin patches,that are dry or red, covered with silvery scales, raised patches ofskin, accompanied by red borders, that may crack and become painful, andthat are usually located on the elbows, knees, trunk, scalp, and hands;skin lesions, including pustules, cracking of the skin, and skinredness; joint pain or aching which may be associated with of arthritis,e.g., psoriatic arthritis.

Treatment for psoriasis often includes a topical corticosteroids,vitamin D analogs, and topical or oral retinoids, or combinationsthereof. In one embodiment, the TNFalpha inhibitor of the invention isadministered in combination with or the presence of one of these commontreatments.

The diagnosis of psoriasis is usually based on the appearance of theskin. Additionally a skin biopsy, or scraping and culture of skinpatches may be needed to rule out other skin disorders. An x-ray may beused to check for psoriatic arthritis if joint pain is present andpersistent.

In one embodiment of the invention, a TNFalpha inhibitor is used totreat psoriasis, including chronic plaque psoriasis, guttate psoriasis,inverse psoriasis, pustular psoriasis, pemphigus vulgaris, erythrodermicpsoriasis, psoriasis associated with inflammatory bowel disease (IBD),and psoriasis associated with rheumatoid arthritis (RA). Specific typesof psoriasis included in the treatment methods of the invention aredescribed in detail below:

a. Chronic Plaque Psoriasis

The formulations and methods of the invention may be used to treatsubjects having chronic plaque psoriasis. Tumor necrosis factor has beenimplicated in the pathophysiology of chronic plaque psoriasis (Asadullahet al. (1999) Br J Dermatol. 141:94). Chronic plaque psoriasis (alsoreferred to as psoriasis vulgaris) is the most common form of psoriasis.Chronic plaque psoriasis is characterized by raised reddened patches ofskin, ranging from coin-sized to much larger. In chronic plaquepsoriasis, the plaques may be single or multiple, they may vary in sizefrom a few millimeters to several centimeters. The plaques are usuallyred with a scaly surface, and reflect light when gently scratched,creating a “silvery” effect. Lesions (which are often symmetrical) fromchronic plaque psoriasis occur all over body, but with predilection forextensor surfaces, including the knees, elbows, lumbosacral regions,scalp, and nails. Occasionally chronic plaque psoriasis can occur on thepenis, vulva and flexures, but scaling is usually absent. Diagnosis ofpatients with chronic plaque psoriasis is usually based on the clinicalfeatures described above. In particular, the distribution, color andtypical silvery scaling of the lesion in chronic plaque psoriasis arecharacteristic of chronic plaque psoriasis.

b. Guttate Psoriasis

The formulations and methods of the invention may be used to treatsubjects having guttate psoriasis. Guttate psoriasis refers to a form ofpsoriasis with characteristic water drop shaped scaly plaques. Flares ofguttate psoriasis generally follow an infection, most notably astreptococcal throat infection. Diagnosis of guttate psoriasis isusually based on the appearance of the skin, and the fact that there isoften a history of recent sore throat.

c. Inverse Psoriasis

The formulations and methods of the invention may be used to treatsubjects having inverse psoriasis. Inverse psoriasis is a form ofpsoriasis in which the patient has smooth, usually moist areas of skinthat are red and inflamed, which is unlike the scaling associated withplaque psoriasis. Inverse psoriasis is also referred to as intertiginouspsoriasis or flexural psoriasis. Inverse psoriasis occurs mostly in thearmpits, groin, under the breasts and in other skin folds around thegenitals and buttocks, and, as a result of the locations ofpresentation, rubbing and sweating can irritate the affected areas.

d. Pustular Psoriasis

The formulations and methods of the invention may be used to treatsubjects having pustular psoriasis. Pustular psoriasis is a form ofpsoriasis that causes pus-filled blisters that vary in size andlocation, but often occur on the hands and feet. The blisters may belocalized, or spread over large areas of the body. Pustular psoriasiscan be both tender and painful, can cause fevers.

e. Other Psoriasis Disorders

Other examples of psoriatic disorders which can be treated with theformulations and methods of the invention include erythrodermicpsoriasis, vulgaris, psoriasis associated with IBD, and psoriasisassociated with arthritis, including rheumatoid arthritis.

2. Pemphigus Vulgaris

The formulations and methods of the invention may be used to treatsubjects having pemphigus vulgaris. Pemphigus vulgaris is a seriousautoimmune systemic dermatologic disease that often affects the oralmucous membrane and skin. The pathogenesis of pemphigus vulgaris isthought to be an autoimmune process that is directed at skin and oralmucous membrane desmosomes. Consequentially, cells do not adhere to eachother. The disorder manifests as large fluid-filled, rupture-pronebullae, and has a distinctive histologic appearance. Anti-inflammatoryagents are the only effective therapy for this disease which has a highmortality rate. Complications that arise in patients suffering frompemphigus vulgaris are intractable pain, interference with nutrition andfluid loss, and infections.

3. Atopic Dermatitis/Eczema

The formulations and methods of the invention may be used to treatsubjects having atopic dermatitis. Atopic dermatitis (also referred toas eczema) is a chronic skin disorder categorized by scaly and itchingplaques. People with eczema often have a family history of allergicconditions like asthma, hay fever, or eczema. Atopic dermatitis is ahypersensitivity reaction (similar to an allergy) which occurs in theskin, causing chronic inflammation. The inflammation causes the skin tobecome itchy and scaly. Chronic irritation and scratching can cause theskin to thicken and become leathery-textured. Exposure to environmentalirritants can worsen symptoms, as can dryness of the skin, exposure towater, temperature changes, and stress.

Subjects with atopic dermatitis can be identified by certain symptoms,which often include intense itching, blisters with oozing and crusting,skin redness or inflammation around the blisters, rash, dry, leatheryskin areas, raw areas of the skin from scratching, and eardischarges/bleeding.

4. Sarcoidosis

The formulations and methods of the invention may be used to treatsubjects having sarcoidosis. Sarcoidosis is a disease in whichgranulomatous inflammation occurs in the lymph nodes, lungs, liver,eyes, skin, and/or other tissues. Sarcoidosis includes cutaneoussarcoidosis (sarcoidosis of the skin) and nodular sarcoidosis(sarcoidosis of the lymph nodes). Patients with sarcoidosis can beidentified by the symptoms, which often include general discomfort,uneasiness, or an ill feeling; fever; skin lesions.

5. Erythema Nodosum

The formulations and methods of the invention may be used to treatsubjects having erythema nodosum. Erythema nodosum refers to aninflammatory disorder that is characterized by tender, red nodules underthe skin, typically on the anterior lower legs. Lesions associated witherythema nodosum often begin as flat, but firm, hot red painful lumps(approximately an inch across). Within a few days the lesions may becomepurplish, and then over several weeks fade to a brownish flat patch.

In some instances, erythema nodosum may be associated with infectionsincluding, streptococcus, coccidioidomycosis, tuberculosis, hepatitis B,syphilis, cat scratch disease, tularemia, yersinia, leptospirosispsittacosis, histoplasmosis, mononucleosis (EBV). In other instances,erythema nodosum may be associated with sensitivity to certainmedications including, oralcontraceptives, penicillin, sulfonamides,sulfones, barbiturates, hydantoin, phenacetin, salicylates, iodides, andprogestin. Erythema nodosum is often associated with other disordersincluding, leukemia, sarcoidosis, rheumatic fever, and ulcerativecolitis.

Symptoms of erythema nodosum usually present themselves on the shins,but lesions may also occur on other areas of the body, including thebuttocks, calves, ankles, thighs and upper extremities. Other symptomsin subjects with erythema nodosum can include fever and malaise.

6. Hidradenitis Suppurativa

The formulations and methods of the invention may be used to treatsubjects having hidradenitis suppurativa. Hidradenitis suppurativarefers to a skin disorder in which swollen, painful, inflamed lesions orlumps develop in the groin and sometimes under the arms and under thebreasts. Hidradenitis suppurativa occurs when apocrine gland outletsbecome blocked by perspiration or are unable to drain normally becauseof incomplete gland development. Secretions trapped in the glands forceperspiration and bacteria into surrounding tissue, causing subcutaneousinduration, inflammation, and infection. Hidradenitis suppurativa isconfined to areas of the body that contain apocrine glands. These areasare the axillae, areola of the nipple, groin, perineum, circumanal, andperiumbilical regions.

7. Lichen Planus

The formulations and methods of the invention may be used to treatsubjects having lichen planus. Tumor necrosis factor has been implicatedin the pathophysiology of lichen planus (Sklavounou et al. (2000) J OralPathol Med. 29:370). Lichen planus refers to a disorder of the skin andthe mucous membranes resulting in inflammation, itching, and distinctiveskin lesions. Lichen planus may be associated with hepatitis C orcertain medications.

8. Sweet's Syndrome

The formulations and methods of the invention may be used to treatsubjects having Sweet's syndrome. Inflammatory cytokines, includingtumor necrosis factor, have been implicated in the pathophysiology ofSweet's syndrome (Reuss-Borst et al. (1993) Br J Haematol. 84:356).Sweet's syndrome, which was described by R. D. Sweet in 1964, ischaracterized by the sudden onset of fever, leukocytosis, and cutaneouseruption. The eruption consists of tender, erythematous, well-demarcatedpapules and plaques which show dense neutrophilic infiltratesmicroscopically. The lesions may appear anywhere, but favor the upperbody including the face. The individual lesions are often described aspseudovesicular or pseudopustular, but may be frankly pustular, bullous,or ulcerative. Oral and eye involvement (conjunctivitis or episcleritis)have also been frequently reported in patients with Sweet's syndrome.Leukemia has also been associated with Sweet's syndrome.

9. Vitiligo

The formulations and methods of the invention may be used to treatsubjects having vitiligo. Vitiligo refers to a skin condition in whichthere is loss of pigment from areas of skin resulting in irregular whitepatches with normal skin texture. Lesions characteristic of vitiligoappear as flat depigmented areas. The edges of the lesions are sharplydefined but irregular. Frequently affected areas in subjects withvitiligo include the face, elbows and knees, hands and feet, andgenitalia.

10. Scleroderma

The formulations and methods of the invention may be used to treatsubjects having scleroderma. Tumor necrosis factor has been implicatedin the pathophysiology of scleroderma (Tutuncu Z et al. (2002) Clin ExpRheumatol. 20(6 Suppl 28):S146-51; Mackiewicz Z et al. (2003) Clin ExpRheumatol. 21(1):41-8; Murota H et al. (2003) Arthritis Rheum.48(4):1117-25). Scleroderma refers to a diffuse connective tissuedisease characterized by changes in the skin, blood vessels, skeletalmuscles, and internal organs. Scleroderma is also referred to as CRESTsyndrome or Progressive systemic sclerosis, and usually affects peoplebetween the ages 30-50. Women are affected more often than men.

The cause of scleroderma is unknown. The disease may produce local orsystemic symptoms. The course and severity of the disease varies widelyin those affected. Excess collagen deposits in the skin and other organsproduce the symptoms. Damage to small blood vessels within the skin andaffected organs also occurs. In the skin, ulceration, calcification, andchanges in pigmentation may occur. Systemic features may includefibrosis and degeneration of the heart, lungs, kidneys andgastrointestinal tract.

Patients suffering from scleroderma exhibit certain clinical features,including, blanching, blueness, or redness of fingers and toes inresponse to heat and cold (Raynaud's phenomenon), pain, stiffness, andswelling of fingers and joints, skin thickening and shiny hands andforearm, esophageal reflux or heartburn, difficulty swallowing, andshortness of breath. Other clinical symptoms used to diagnosescleroderma include, an elevated erythrocyte sedimentation rate (ESR),an elevated rheumatoid factor (RF), a positive antinuclear antibodytest, urinalysis that shows protein and microscopic blood, a chest X-raythat may show fibrosis, and pulmonary function studies that showrestrictive lung disease.

11. Nail Disorders

The formulations and methods of the invention may be used to treatsubjects having a nail disorder. Nail disorders include any abnormalityof the nail Specific nail disorders include, but are not limited to,pitting, koilonychia, Beau's lines, spoon nails, onycholysis, yellownails, pterygium (seen in lichen planus), and leukonychia. Pitting ischaracterized by the presence of small depressions on the nail surface.Ridges or linear elevations can develop along the nail occurring in a“lengthwise” or “crosswise” direction. Beau's lines are lineardepressions that occur “crosswise” (transverse) in the fingernail.Leukonychia describes white streaks or spots on the nails. Koilonychiais an abnormal shape of the fingernail where the nail has raised ridgesand is thin and concave Koilonychia is often associated with irondeficiency.

Nail disorders which can be treated with the TNFalpha antibody of theinvention also include psoriatic nails. Psoriatic nails include changesin nails which are attributable to psoriasis. In some instancespsoriasis may occur only in the nails and nowhere else on the body.Psoriatic changes in nails range from mild to severe, generallyreflecting the extent of psoriatic involvement of the nail plate, nailmatrix, i.e., tissue from which the nail grows, nail bed, i.e., tissueunder the nail, and skin at the base of the nail Damage to the nail bedby the pustular type of psoriasis can result in loss of the nail Nailchanges in psoriasis fall into general categories that may occur singlyor all together. In one category of psoriatic nails, the nail plate isdeeply pitted, probably due to defects in nail growth caused bypsoriasis. IN another category, the nail has a yellow to yellow-pinkdiscoloration, probably due to psoriatic involvement of the nail bed. Athird subtype of psoriatic nails are characterized by white areas whichappear under the nail plate. The white areas are actually air bubblesmarking spots where the nail plate is becoming detached from the nailbed. There may also be reddened skin around the nail A fourth categoryis evidenced by the nail plate crumbling in yellowish patches, i.e.,onychodystrophy, probably due to psoriatic involvement in the nailmatrix. A fifth category is characterized by the loss of the nail in itsentirety due to psoriatic involvement of the nail matrix and nail bed.

The formulations and methods of the invention may also be used to treatnail disorders often associated with lichen planus. Nails in subjectswith lichen planus often show thinning and surface roughness of the nailplate with longitudinal ridges or pterygium.

The formulations and methods of the invention may be used to treat naildisorders, such as those described herein. Often nail disorders areassociated with skin disorders. In one embodiment, the inventionincludes a method of treatment for nail disorders with a TNFalphaantibody. In another embodiment, the nail disorder is associated withanother disorder, including a skin disorder such as psoriasis. Inanother embodiment, the disorder associated with a nail disorder isarthritis, including psoriatic arthritis.

12. Other Skin and Nail Disorders

The formulations and methods of the invention may be used to treat otherskin and nail disorders, such as chronic actinic dermatitis, bullouspemphigoid, and alopecia areata. Chronic actinic dermatitis (CAD) isalso referred to as photosensitivity dermatitis/actinic reticuloidsyndrome (PD/AR). CAD is a condition in which the skin becomes inflamed,particularly in areas that have been exposed to sunlight or artificiallight. Commonly, CAD patients have allergies to certain substances thatcome into contact with their skin, particularly various flowers, woods,perfumes, sunscreens and rubber compounds. Bullous pemphigoid refers toA skin disorder characterized by the formation of large blisters on thetrunk and extremities. Alopecia areata refers to hair loss characterizedby round patches of complete baldness in the scalp or beard.

K. Metabolic Disorders

The formulations and methods of the invention may be used to treat ametabolic disease. TNFα has been implicated in the pathophysiology of awide variety of disorders, including metabolic disorders, such asdiabetes and obesity (Spiegelman and Hotamisligil (1993) Cell 73:625;Chu et al. (2000) Int J Obes Relat Metab Disord. 24:1085; Ishii et al.(2000) Metabolism. 49:1616).

Metabolic disorders affect how the body processes substances needed tocarry out physiological functions. A number of metabolic disorders ofthe invention share certain characteristics, i.e. they are associatedthe insulin resistance, lack of ability to regulate blood sugar, weightgain, and increase in body mass index. Examples of metabolic disordersinclude diabetes and obesity. Examples of diabetes include type 1diabetes mellitus, type 2 diabetes mellitus, diabetic neuropathy,peripheral neuropathy, diabetic retinopathy, diabetic ulcerations,retinopathy ulcerations, diabetic macrovasculopathy, and obesity.Examples of metabolic disorders which can be treated with theformulations and methods of the invention are described in more detailbelow:

1. Diabetes

The formulations and methods of the invention may be used to treatdiabetes. Tumor necrosis factor has been implicated in thepathophysiology of diabetes. (see e.g., Navarro J. F., Mora C., Maca, AmJ Kidney Dis. 2003 July; 42(1):53-61; Daimon M et al., Diabetes Care.2003 July; 26(7):2015-20; Zhang M et al., J Tongji Med Univ. 1999;19(3):203-5, Barbieri M et al., Am J Hypertens. 2003 July;16(7):537-43.) For example, TNFα is implicated in the pathophysiologyfor insulin resistance. It has been found that serum TNF levels inpatients with gastrointestinal cancer correlates with insulin resistance(see e.g., McCall, J. et al. Br. J. Surg. 1992; 79: 1361-3).

Diabetes includes the two most common types of the disorder, namely typeI diabetes and type II diabetes, which both result from the body'sinability to regulate insulin. Insulin is a hormone released by thepancreas in response to increased levels of blood sugar (glucose) in theblood.

The term “type 1 diabetes,” as used herein, refers to a chronic diseasethat occurs when the pancreas produces too little insulin to regulateblood sugar levels appropriately. Type 1 diabetes is also referred to asinsulin-dependent diabetes mellitus, IDMM, juvenile onset diabetes, anddiabetes—type I. Type 1 diabetes represents is the result of aprogressive autoimmune destruction of the pancreatic β-cells withsubsequent insulin deficiency.

The term “type 2 diabetes,” refers to a chronic disease that occurs whenthe pancreas does not make enough insulin to keep blood glucose levelsnormal, often because the body does not respond well to the insulin.Type 2 diabetes is also referred to as noninsulin-dependent diabetesmellitus, NDDM, and diabetes—type II

Diabetes is can be diagnosed by the administration of a glucosetolerance test. Clinically, diabetes is often divided into several basiccategories. Primary examples of these categories include, autoimmunediabetes mellitus, non-insulin-dependent diabetes mellitus (type 1NDDM), insulin-dependant diabetes mellitus (type 2 IDDM), non-autoimmunediabetes mellitus, non-insulin-dependant diabetes mellitus (type 2NIDDM), and maturity-onset diabetes of the young (MODY). A furthercategory, often referred to as secondary, refers to diabetes broughtabout by some identifiable condition which causes or allows a diabeticsyndrome to develop. Examples of secondary categories include, diabetescaused by pancreatic disease, hormonal abnormalities, drug- orchemical-induced diabetes, diabetes caused by insulin receptorabnormalities, diabetes associated with genetic syndromes, and diabetesof other causes. (see e.g., Harrison's (1996) 14^(th) ed., New York,McGraw-Hill).

Diabetes manifests itself in the foregoing categories and can causeseveral complications that are discussed in the following sections.Accordingly, the antibody, or antigen-binding fragment thereof, of theinvention can be used to treat diabetes. In one embodiment, the TNFαantibody, or antigen-binding fragment thereof, of the invention is usedto treat diabetes associated with the above identified categories.

Diabetes is often treated with diet, insulin dosages, and variousmedications described herein. Accordingly, the formulations of theinvention may also be administered in combination with agents commonlyused to treat metabolic disorders and pain commonly associated withdiabetes.

Diabetes manifests itself in many complications and conditionsassociated with diabetes, including the following categories:

a. Diabetic Neuropathy and Peripheral Neuropathy

The formulations and methods of the invention may be used to treatdiabetic neuropathy or peripheral neuropathy. Tumor necrosis factor hasbeen implicated in the pathophysiology of diabetic neuropathy andperipheral neuropathy. (See Benj afield et al. (2001) Diabetes Care.24:753; Qiang, X. et al. (1998) Diabetologia. 41:1321-6; Pfeiffer et al.(1997) Horm Metab Res. 29:111).

The term “neuropathy,” also referred to as nerve damage-diabetic, asused herein, refers to a common complication of diabetes in which nervesare damaged as a result of hyperglycemia (high blood sugar levels). Avariety of diabetic neuropathies are recognized, such as distalsensorimotror polyneuropathy, focal motor neuropathy, and autonomicneuropathy.

The term “peripheral neuropathy,” also known as peripheral neuritis anddiabetic neuropathy, as used herein, refers to the failure of the nervesto carry information to and from the brain and spinal cord. Peripheralneuropathy produces symptoms such as pain, loss of sensation, and theinability to control muscles. In some cases, the failure of nerves tocontrol blood vessels, intestinal function, and other organs results inabnormal blood pressure, digestion, and loss of other basic involuntaryprocesses. Peripheral neuropathy may involve damage to a single nerve ornerve group (mononeuropathy) or may affect multiple nerves(polyneuropathy).

Neuropathies that affect small myelinated and unmyelinated fibers of thesympathetic and parasympathetic nerves are known as “peripheralneuropathies.” Furthermore, the related disorder of peripheralneuropathy, also known as peripheral neuritis and diabetic neuropathy,refers to the failure of the nerves to carry information to and from thebrain and spinal cord. This produces symptoms such as pain, loss ofsensation, and the inability to control muscles. In some cases, failureof nerves controlling blood vessels, intestinal function, and otherorgans results in abnormal blood pressure, digestion, and loss of otherbasic involuntary processes. Peripheral neuropathy may involve damage toa single nerve or nerve group (mononeuropathy) or may affect multiplenerves (polyneuropathy).

The term “diabetic neuropathy” refers to a common complication ofdiabetes in which nerves are damaged as a result of hyperglycemia (highblood sugar levels). Diabetic neuropathy is also referred to asneuropathy and nerve damage-diabetic. A variety of diabetic neuropathiesare recognized, such as distal sensorimotror polyneuropathy, focal motorneuropathy, and autonomic neuropathy.

b. Diabetic Retinopathy

The formulations and methods of the invention may be used to treatdiabetic retinopathy. Tumor necrosis factor has been implicated in thepathophysiology of diabetic retinopthy (Scholz et al. (2003) TrendsMicrobiol. 11:171). The term “diabetic retinopathy” as used herein,refers to progressive damage to the eye's retina caused by long-termdiabetes. Diabetic retinopathy, includes proliferative retinopathy.Proliferative neuropathy in turn includes includes neovascularization,pertinal hemmorrhave and retinal detachement.

In advanced retinopathy, small vessels proliferate on the surface of theretina. These blood vessels are fragile, tend to bleed and can causeperetinal hemorrhages. The hemorrhage can obscure vision, and as thehemorrhage is resorbed fibrous tissue forms predisposing to retinaldetachments and loss of vision. In addition, diabetic retinopathyincludes prolferative retinopathy which includes neovascularization,pertinal hemmorrhave and retinal detachement. Daibetic retinopathy alsoincludes “background retinopathy” which involves changes occuring withthe layers of the retina.

c. Diabetic Ulcerations and Retinopathy Ulcerations

The formulations and methods of the invention may be used to treatdiabetic ulcerations or retinopathy ulcerations. Tumor necrosis factorhas been implicated in the pathophysiology of diabetic ulcerations, (seeLee et al. (2003) Hum Immunol. 64:614; Navarro et al. (2003) Am J KidneyDis. 42:53; Daimon et al (2003) Diabetes Care. 26:2015; Zhang et al.(1999) J Tongji Med Univ. 19:203; Barbieri et al. (2003) Am J Hypertens.16:537; Venn et al. (1993) Arthritis Rheum. 36:819; Westacott et al.(1994) J Rheumatol. 21:1710).

The term “diabetic ulcerations,” as used herein, refers to an ulcerwhich results as a complication of diabetes. An ulcer is a crater-likelesion on the skin or mucous membrane caused by an inflammatory,infectious, malignant condition, or metabolic disorder. Typicallydiabetic ulcers can be found on limbs and extremeties, more typicallythe feet. These ulcers, caused by diabetic conditions, such as neurapthyand a vacualr insuffciency, can lead to ischemia and poor wound healing.More extensive ulcerations may progress to ostemyelitis. Onceostemyelitis develops, it may be dificulte to eradicate with antiboticsalonda nd amputation maybe necessary.

The term “retinopathy ulcerations,” as used herein refers to an ulcerwhich causes or results in damages to the eye and the eye's retina.Retinopathy ulcerations may include conditions such has retinoathichemmorages.

d. Diabetic Macrovasculopathy

The formulations and methods of the invention may be used to treatdiabetic macrovasculopathy. Tumor necrosis factor has been implicated inthe pathophysiology of diabetic macrovasculopathy (Devaraj et al. (2000)Circulation. 102:191; Hattori Y et al. (2000) Cardiovasc Res. 46:188;Clausell N et al. (1999) Cardiovasc Pathol. 8:145). The term “diabeticmacrovasculopathy,” also referred to as “macrovascular disease,” as usedherein, refers to a disease of the blood vessels that results fromdiabetes. Diabetic macrovasculopathy complication occurs when, forexample, fat and blood clots build up in the large blood vessels andstick to the vessel walls. Diabetic macrovasculopathies include diseasessuch as coronary disease, cerebrovascular disease, and peripheralvascular disease, hyperglycaemia and cardiovascular disease, andstrokes.

2. Obesity

The formulations and methods of the invention may be used to treatobesity. Tumor necrosis factor has been implicated in thepathophysiology of obesity (see e.g., Pihlajamaki J et al. (2003) ObesRes. 11:912; Barbieri et al. (2003) Am J Hypertens. 16:537; Tsuda et al.(2003) J Nutr. 133:2125). Obesity increases a person's risk of illnessand death due to diabetes, stroke, coronary artery disease,hypertension, high cholesterol, and kidney and gallbladder disorders.Obesity may also increase the risk for some types of cancer, and may bea risk factor for the development of osteoarthritis and sleep apnea.Obesity can be treated with the antibody of the invention alone or incombination with other metabolic disorders, including diabetes.

L. Vasculitides

The formulations and methods of the invention may be used to treat asubject having a vasculitis. TNFα has been implicated in thepathophysiology of a variety of vasculitides, (see e.g., Deguchi et al.(1989) Lancet. 2:745). As used herein, the term “a vasculitis in whichTNFα activity is detrimental” is intended to include vasculitis in whichthe presence of TNFα in a subject suffering from the disorder has beenshown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Such disorders may be evidenced, for example,by an increase in the concentration of TNFα in a biological fluid of asubject suffering from the disorder (e.g., an increase in theconcentration of TNFα in serum, plasma, synovial fluid, etc. of thesubject), which can be detected, for example, using an anti-TNFαantibody as described above.

There are numerous examples of vasculitides in which TNFα activity isdetrimental, including Behcet's disease. The use of the formulations andmethods of the invention in the treatment of specific vasculitides arediscussed further below. In certain embodiments, the antibody, orantibody portion, is administered to the subject in combination withanother therapeutic agent, as described below

The formulations and methods of the invention be used to treatvasculitis in which TNFα activity is detrimental, wherein inhibition ofTNFα activity is expected to alleviate the symptoms and/or progressionof the vasculitis or to prevent the vasculitis. Subjects suffering fromor at risk of developing vasculitis can be identified through clinicalsymptoms and tests. For example, subjects with vasculitides oftendevelop antibodies to certain proteins in the cytoplasm of neutrophils,antineutrophil cytoplasmic antibodies (ANCA). Thus, in some instances,vasculitides may be evidenced by tests (e.g., ELISA), which measure ANCApresence.

Vasculitis and its consequences may be the sole manifestation of diseaseor it may be a secondary component of another primary disease.Vasculitis may be confined to a single organ or it may simultaneouslyaffect several organs. and depending on the syndrome, arteries and veinsof all sizes can be affected. Vasculitis can affect any organ in thebody.

In vasculitis, the vessel lumen is usually compromised, which isassociated with ischemia of the tissues supplied by the involved vessel.The broad range of disorders that may result from this process is due tothe fact that any type, size and location of vessel (e.g., artery, vein,arteriole, venule, capillary) can be involved. Vasculitides aregenerally classified according to the size of the affected vessels, asdescribed below. It should be noted that some small and large vesselvasculitides may involve medium-sized arteries; but large andmedium-sized vessel vasculitides do not involve vessels smaller thanarteries. Large vessel disease includes, but is not limited to, giantcell arteritis, also known as temporal arteritis or cranial arteritis,polymyalgia rheumatica, and Takayasu's disease or arteritis, which isalso known as aortic arch syndrome, young female arteritis and Pulselessdisease. Medium vessel disease includes, but is not limited to, classicpolyarteritis nodosa and Kawasaki's disease, also known as mucocutaneouslymph node syndrome. Non-limiting examples of small vessel disease areBehcet's Syndrome, Wegner's granulomatosis, microscopic polyangitis,hypersensitivity vasculitis, also known as cutaneous vasculitis, smallvessel vasculitis, Henoch-Schonlein purpura, allergic granulamotosis andvasculitis, also known as Churg Strauss syndrome. Other vasculitidesinclude, but are not limited to, isolated central nervous systemvasculitis, and thromboangitis obliterans, also known as Buerger'sdisease. Classic Polyarteritis nodosa (PAN), microscopic PAN, andallergic granulomatosis are also often grouped together and are calledthe systemic necrotizing vasculitides. A further description ofvasculitis is described below:

1. Large Vessel Vasculitis

In one embodiment, the formulations and methods of the invention areused to treat subjects who have large vessel vasculitis. The term “largevessel(s)” as used herein, refers to the aorta and the largest branchesdirected toward major body regions. Large vessels include, for example,the aorta, and its branches and corresponding veins, e.g., thesubclavian artery; the brachiocephalic artery; the common carotidartery; the innonimate vein; internal and external jugular veins; thepulmonary arteries and veins; the venae cavae; the renal arteries andveins; the femoral arteries and veins; and the carotid arteries.Examples of large vessel vasculitides are described below.

a. Giant Cell Arteritis (GCA)

The formulations and methods of the invention may be used to treat giantcell arteritis. Tumor necrosis factor has been implicated in thepathophysiology of giant cell arteritis (Sneller, M. C. (2002) Cleve.Clin. J. Med. 69:SII40-3; Schett, G., et al. (2002) Ann. Rheum. Dis.61:463). Giant cell arteritis (GCA), refers to a vasculitis involvinginflammation and damage to blood vessels, particularly the large ormedium arteries that branch from the external carotid artery of theneck. GCA is also referred to as temporal arteritis or cranialarteritis, and is the most common primary vasculitis in the elderly. Italmost exclusively affects individuals over 50 years of age, however,there are well-documented cases of patients 40 years and younger. GCAusually affects extracranial arteries. GCA can affect the branches ofthe carotid arteries, including the temporal artery. GCA is also asystemic disease which can involve arteries in multiple locations.

Histopathologically, GCA is a panarteritis with inflammatory mononuclearcell infiltrates within the vessel wall with frequent Langhans typegiant cell formation. There is proliferation of the intima,granulomatous inflammation and fragmentation of the internal elasticlamina. The pathological findings in organs is the result of ischemiarelated to the involved vessels.

Patients suffering from GCA exhibit certain clinical symptoms, includingfever, headache, anemia and high erythrocyte sedimentation rate (ESR).Other typical indications of GCA include jaw or tongue claudication,scalp tenderness, constitutional symptoms, pale optic disc edema(particularly ‘chalky white’ disc edema), and vision disturbances. Thediagnosis is confirmed by temporal artery biopsy.

b. Polymyalgia Rheumatica

The formulations and methods of the invention may be used to treatpolymyalgia rheumatica. Tumor necrosis factor has been implicated in thepathophysiology of polymyalgia rheumatica (Straub, R. H., et al. (2002)Rheumatology (Oxford) 41:423; Uddhammar, A., et al. (1998) Br. J.Rheumatol. 37:766). Polymyalgia rheumatica refers to a rheumaticdisorder that is associated with moderate to severe muscle pain andstiffness in the neck, shoulder, and hip, most noticeable in themorning. IL-6 and IL-1β expression has also been detected in a majorityof the circulating monocytes in patients with the polymyalgiarheumatica. Polymyalgia rheumatica may occur independently, or it maycoexist with or precede GCA, which is an inflammation of blood vessels.

c. Takayasu's Arteritis

The formulations and methods of the invention may be used to treatTakayasu's arteritis. Tumor necrosis factor has been implicated in thepathophysiology of Takayasu's arteritis (Kobayashi, Y. and Numano, F.(2002) Intern. Med. 41:44; Fraga, A. and Medina F. (2002) Curr.Rheumatol. Rep. 4:30). Takayasu's arteritis refers to a vasculitischaracterized by an inflammmation of the aorta and its major branches.Takayasu's arteritis (also known as Aortic arch syndrome, young femalearteritis and Pulseless disease) affects the thoracic and abdominalaorta and its main branches or the pulmonary arteries. Fibroticthickening of the aortic wall and its branches (e.g., carotid,inominate, and subclavian arteries) can lead to reduction of lumen sizeof vessels that arise from the aortic arch. This condition alsotypically affects the renal arteries.

Takayasu's arteritis primarily affects young women, usually aged 20-40years old, particularly of Asian descent, and may be manifested bymalaise, arthralgias and the gradual onset of extremity claudication.Most patients have asymmetrically reduced pulses, usually along with ablood pressure differential in the arms. Coronary and/or renal arterystenosis may occur.

The clinical features of Takayasu's arteritis may be divided into thefeatures of the early inflammatory disease and the features of the laterdisease. The clinical features of the early inflammatory stage ofTakayasu's disease are: malaise, low grade fever, weight loss, myalgia,arthralgia, and erythema multiforme. Later stages of Takayasu's diseaseare characterised by fibrotic stenosis of arteries and thrombosis. Themain resulting clinical features are ischaemic phenomena, e.g. weak andasymmetrical arterial pulses, blood pressure discrepancy between thearms, visual disturbance, e.g. scotomata and hemianopia, otherneurological features including vertigo and syncope, hemiparesis orstroke. The clinical features result from ischaemia due to arterialstenosis and thrombosis.

2. Medium Vessel Disease

The formulations and methods of the invention may be used to treatsubjects who have medium vessel vasculitis. The term “medium vessel(s)”is used to refer to those blood vessels which are the main visceralarteries. Examples of medium vessels include the mesenteric arteries andveins, the iliac arteries and veins, and the maxillary arteries andveins. Examples of medium vessel vasculitides are described below.

a. Polyarteritis Nodosa

The formulations and methods of the invention may be used to treatpolyarteritis nodosa. Tumor necrosis factor has been implicated in thepathophysiology of polyarteritis nodosa (DiGirolamo, N., et al. (1997)J. Leukoc. Biol. 61:667). Polyarteritis nodosa, or periarteritis nodosarefers to vasculitis which is a serious blood vessel disease in whichsmall and medium-sized arteries become swollen and damaged because theyare attacked by rogue immune cells. Polyarteritis nodosa usually affectsadults more frequently than children. It damages the tissues supplied bythe affected arteries because they don't receive enough oxygen andnourishment without a proper blood supply.

Symptoms which are exhibited in patients with polyarteritis nodosagenerally result from damage to affected organs, often the skin, heart,kidneys, and nervous system. Generalized symptoms of polyarteritisnodosa include fever, fatigue, weakness, loss of appetite, and weightloss. Muscle aches (myalgia) and joint aches (arthralgia) are common.The skin of subjects with polyarteritis nodosa may also show rashes,swelling, ulcers, and lumps (nodular lesions).

Classic PAN (polyarteritis nodosa) is a systemic arteritis of small tomedium muscular arteritis in which involvement of renal and visceralarteries is common Abdominal vessels have aneurysms or occlusions in 50%of PAN patients. Classic PAN does not involve the pulmonary arteriesalthough the bronchial vessels may be involved. Granulomas, significanteosinophilia and an allergic diathesis are not part of the syndrome.Although any organ system may be involved, the most commonmanifestations include peripheral neuropathy, mononeuritis multiplex,intestinal ischemia, renal ischemia, testicular pain and livedoreticularis.

b. Kawasaki's Disease

The formulations and methods of the invention may be used to treatKawasaki's disease. Tumor necrosis factor has been implicated in thepathophysiology of Kawasaki's disease (Sundel, R. P. (2002) Curr.Rheumatol. Rep. 4:474; Gedalia, A. (2002) Curr. Rheumatol. Rep. 4:25).Although the cause of Kawasaki's disease is unknown, it is associatedwith acute inflammation of the coronary arteries, suggesting that thetissue damage associated with this disease may be mediated byproinflammatory agents such as TNFα. Kawasaki's disease refers to avasculitis that affects the mucus membranes, lymph nodes, lining of theblood vessels, and the heart. Kawasaki's disease is also often referredto as mucocutaneous lymph node syndrome, mucocutaneous lymph nodedisease, and infantile polyarteritis. Subjects afflicted with Kawasaki'sdisease develop vasculitis often involving the coronary arteries whichcan lead to myocarditis and pericarditis. Often as the acuteinflammation diminishes, the coronary arteries may develop aneurysm,thrombosis, and lead to myocardial infarction.

Kawasaki's disease is a febrile systemic vasculitis associated withedema in the palms and the soles of the feet, with enlargement ofcervical lymph nodes, cracked lips and “strawberry tongue”. Although theinflammatory response is found in vessels throughout the body, the mostcommon site of end-organ damage is the coronary arteries. Kawasaki'sDisease predominantly affects children under the age of 5. The highestincidence is in Japan but is becoming increasingly recognized in theWest and is now the leading cause of acquired heart disease in USchildren. The most serious complication of Kawasaki disease is coronaryarteritis and aneurysm formation that occurs in a third of untreatedpatients.

3. Small Vessel Disease

The formulations and methods of the invention may be used to treat smallvessel disease. In one embodiment, the TNFα antibody of the invention isused to treat subjects who have small vessel vasculitis. The term “smallvessel(s)” is used to refer to arterioles, venules and capillaries.Arterioles are arteries that contain only 1 or 2 layers of sooth musclecells and are terminal to and continuous with the capillary network.Venules carry blood from the capillary network to veins and capillariesconnect arterioles and venules. Examples of small vessel vasculitidesare described below.

a. Behcet's Disease

The formulations and methods of the invention may be used to treatBehcet's disease. Tumor necrosis factor has been implicated in thepathophysiology of Behcet's disease (Sfikakis, P. P. (2002) Ann. Rheum.Dis. 61:ii51-3; Dogan, D. and Farah, C. (2002) Oftalmologia. 52:23).Behcet's disease is a chronic disorder that involves inflammation ofblood vessels throughout the body. Behcet's disease may also causevarious types of skin lesions, arthritis, bowel inflammation, andmeningitis (inflammation of the membranes of the brain and spinal cord).As a result of Behcet's disease, the subject with the disorder may haveinflammation in tissues and organs throughout the body, including thegastrointestinal tract, central nervous system, vascular system, lungs,and kidneys. Behcet's disease is three times more common in males thanfemales and is more common in the east Mediterranean and Japan.

b. Wegener's Granulomatosis

The formulations and methods of the invention may be used to treatWegener's granulomatosis. Tumor necrosis factor has been implicated inthe pathophysiology of Wegener's granulomatosis (Marquez, J., et al.(2003) Curr. Rheumatol. Rep. 5:128; Harman, L. E. and Margo, C. E.(1998) Surv. Ophthalmol. 42:458). Wegener's granulomatosis refers to avasculitis that causes inflammation of blood vessels in the upperrespiratory tract (nose, sinuses, ears), lungs, and kidneys. Wegener'sgranulomatosis is also referred to as midline granulomatosis. Wegener'sgranulomatosis includes a granulomatous inflammation involving therespiratory tract, and necrotizing vasculitis affecting small tomedium-sized vessels. Subjects who have Wegener's granulomatosis oftenalso have arthritis (joint inflammation). Glomerulonephritis may also bepresent in affected subjects, but virtually any organ may be involved.

c. Churg-Strauss Syndrome

The formulations and methods of the invention may be used to treatChurg-Strauss syndrome. Tumor necrosis factor has been implicated in thepathophysiology of Churg-Strauss syndrome (Gross, W. L (2002) Curr.Opin. Rheumatol. 14:11; Churg, W. A. (2001) Mod. Pathol. 14:1284).Churg-Strauss syndrome refers to a vasculitis that is systemic and showsearly manifestation signs of asthma and eosinophilia. Churg-Strausssyndrome is also referred to as allergic granulomatosis and angiitis,and occurs in the setting of allergic rhinitis, asthma and eosinophilia.Sinusitis and pulmonary infiltrates also occur in Churg-Strausssyndrome, primarily affecting the lung and heart. Peripheral neuropathy,coronary arteritis and gastrointestinal involvement are common.

M. Other Diseases

The formulations and methods of the invention may be used to treatvarious other disorders in which TNFalpha activity is detrimental.Examples of other diseases and disorders in which TNFalpha activity hasbeen implicated in the pathophysiology, and thus which can be treatedusing an antibody, or antibody portion, of the invention, includeinflammatory bone disorders and bone resorption disease (see e.g.,Bertolini. D. R., et al. (1986) Nature 319:516-518; Konig, A. et al.(1988) J. Bone Miner. Res. 3:621-627; Lerner, U. H. and Ohlin, A. (1993)J. Bone Miner. Res. 8:147-155; and Shanlar. G. and Stem, P. H. (1993)Bone 14:871-876), hepatitis, including alcoholic hepatitis (see e.g.,McClain, C. J. and Cohen, D. A. (1989) Hepatology 9:349-351; Felver, M.E., el al. (1990) Alcohol. Clin. Exp. Res. 14:255-259; and Hansen, J.,el al. (1994) Hepatology 20:461-474), viral hepatitis (Sheron, N., etal. (1991) J. Hepatol. 12:241-245; and Hussain, M. J., et al. (1994) J.Clin. Pathol. 47:1112-1115), and fulminant hepatitis; coagulationdisturbances (see e.g., van der Poll, T., el al. (1990) N. Engl. J. Med.322:1622-1627; and van der Poll, T., et al. (1991) Prog. Clin. Biol.Res. 367:55-60), bums (see eg., Giroir, B. P., el al. (1994) Am. J.Physiol. 267:H 118-124; and Liu. X. S., el al. (1994) Burns 20:40-44),reperfusion injury (see e.g., Scales. W. E., et al. (1994) Am. JPhysiol. 267:G1122-1127; Serrick, C., el al. (1994) Transplantation58:1158-1162; and Yao, Y. M., et al. (1995) Resuscitation 29:157-168),keloid formation (see e.g., McCauley, R. L., et al. (1992) J. Clin.Immunol. 12:300-308), scar tissue formation; pyrexia; periodontaldisease; obesity and radiation toxicity.

Examples of other disorders that may be treated with the formulationsand methods of the invention are described in US20040126372 and U.S.Pat. No. 6,258,562, each of which is incorporated by reference herein.

In one embodiment, the formulation and methods of the invention are usedto treat rheumatoid arthritis, psoriatic arthritis, or ankylosingspondylitis. The formulation of the invention comprising an isolatedhuman TNF alpha antibody, or antigen-binding portion thereof, (e.g.,adalimumab), may be administered to a human subject according to adosing scheme and dose amount effective for treating rheumatoidarthritis, psoriatic arthritis, or ankylosing spondylitis. In oneembodiment, a dose of about 40 mg of a human TNF alpha antibody, orantigen-binding portion thereof, (e.g., adalimumab) (e.g., 0.4 mL of a100 mg/mL formulation of the invention) in the formulation of theinvention is administered to a human subject every other week for thetreatment of rheumatoid arthritis, psoriatic arthritis, or ankylosingspondylitis. In one embodiment, a dose of about 80 mg of a human TNFalpha antibody, or antigen-binding portion thereof, (e.g., adalimumab)(e.g., 0.8 mL of a 100 mg/mL formulation of the invention) in theformulation of the invention is administered to a human subject monthlyfor the treatment of rheumatoid arthritis, psoriatic arthritis, orankylosing spondylitis. In one embodiment, the formulation isadministered subcutaneously, every other week (also referred to asbiweekly, see methods of administration described in US20030235585,incorporated by reference herein) for the treatment of rheumatoidarthritis, ankylosing spondylitis, or psoriatic arthritis. In oneembodiment, the formulation is administered subcutaneously, monthly forthe treatment of rheumatoid arthritis, ankylosing spondylitis, orpsoriatic arthritis.

In one embodiment, the formulation of the invention is used to treatCrohn's disease or ulcerative colitis. The formulation of the inventioncomprising an isolated human TNF alpha antibody, or antigen-bindingportion thereof, (e.g., adalimumab), may be administered to a humansubject according to a dosing scheme and dose amount effective fortreating Crohn's disease. In one embodiment, a dose of about 160 mg of ahuman TNF alpha antibody, or antigen-binding portion thereof, (e.g.,adalimumab) (e.g., 1.6 mL of a 100 mg/mL formulation of the invention)in the formulation of the invention is administered to a human subjectinitially at about day 1, followed by a subsequent dose of 80 mg of theantibody (e.g., 0.8 mL of a 100 mg/mL formulation of the invention) twoweeks later, followed by administration of about 40 mg (e.g., 0.4 mL ofa 100 mg/mL formulation of the invention) every other week for thetreatment of Crohn's disease. In one embodiment, the formulation isadministered subcutaneously, according to a multiple variable doseregimen comprising an induction dose(s) and maintenance dose(s) (see,for example, U.S. Patent Publication Nos. US20060009385 andUS20090317399, incorporated by reference herein) for the treatment ofCrohn's disease or ulcerative colitis, each of which are incorporated byreference herein) for the treatment of Crohn's disease or ulcerativecolitis. In one embodiment, the formulation is administeredsubcutaneously, biweekly or monthly for the treatment of Crohn's diseaseor ulcerative colitis. In one embodiment, a dose of about 80 mg of ahuman TNF alpha antibody, or antigen-binding portion thereof, (e.g.,adalimumab) (e.g., 0.8 mL of a 100 mg/mL formulation of the invention)in the formulation of the invention is administered to a human subjectmonthly for the treatment of Crohn's disease or ulcerative colitis.

In one embodiment, the formulation of the invention is used to treatpsoriasis. The formulation of the invention comprising an isolated humanTNF alpha antibody, or antigen-binding portion thereof, (e.g.,adalimumab), may be administered to a human subject according to adosing scheme and dose amount effective for treating psoriasis. In oneembodiment, an initial dose of about 80 mg of a human TNF alphaantibody, or antigen-binding portion thereof, (e.g., adalimumab) (e.g.,0.8 mL of a 100 mg/mL formulation of the invention) in the formulationof the invention is administered to a human subject, followed by asubsequent dose of 40 mg of the antibody (e.g., 0.4 mL of a 100 mg/mLformulation of the invention) every other week starting one week afterthe initial dose. In one embodiment, the formulation is administeredsubcutaneously, according to a multiple variable dose regimen comprisingan induction dose(s) and maintenance dose(s) (see, for example, US20060009385 and WO 2007/120823, each of which are incorporated byreference herein) for the treatment of psoriasis In one embodiment, theformulation is administered subcutaneously, biweekly or monthly for thetreatment of psoriasis. In one embodiment, a dose of about 80 mg of ahuman TNF alpha antibody, or antigen-binding portion thereof, (e.g.,adalimumab) (e.g., 0.8 mL of a 100 mg/mL formulation of the invention)in the formulation of the invention is administered to a human subjectmonthly for the treatment of psoriasis.

In one embodiment, the formulation of the invention is used to treatjuvenile idiopathic arthritis (JIA). The formulation of the inventioncomprising an isolated human TNF alpha antibody, or antigen-bindingportion thereof, (e.g., adalimumab), may be administered to a humansubject according to a dosing scheme and dose amount effective fortreating JIA. In one embodiment, 20 mg of a human TNF alpha antibody, orantigen-binding portion thereof, in the formulation of the invention(e.g., 0.2 mL of a 100 mg/mL formulation of the invention) isadministered to a subject weighing 15 kg (about 33 lbs) to less than 30kg (66 lbs) every other week for the treatment of JIA. In anotherembodiment, 40 mg of a human TNF alpha antibody, or antigen-bindingportion thereof, in the formulation of the invention (e.g., 0.4 mL of a100 mg/mL formulation of the invention) is administered to a subjectweighing more than or equal to 30 kg (66 lbs) every other week for thetreatment of JIA. In one embodiment, the formulation is administeredsubcutaneously, according to a weight-based fixed dose (see, forexample, U.S. Patent Publication No. 20090271164, incorporated byreference herein) for the treatment of JIA. In one embodiment, theformulation is administered subcutaneously biweekly or monthly for thetreatment of JIA

In one embodiment, an isolated human TNF alpha antibody, orantigen-binding portion thereof, (e.g., adalimumab), may be administeredto a human subject for treatment of a disorder associated withdetrimental TNFa activity according to a monthly dosing schedule,whereby the antibody is administered once every month or once every fourweeks. As described above, examples of disorders that may be treatedaccording to a monthly dosing schedule using the formulations andmethods of the invention include, but are not limited to, rheumatoidarthritis, ankylosing spondylitis, JIA, psoriasis, Crohn's disease,ulcerative colitis, hidradenitis suppurativa, giant cell arteritis,Behcet's disease, sarcoidosis, diabetic retinopathy, or psoriaticarthritis. Thus, the formulation of the invention comprising an isolatedhuman TNF alpha antibody, or antigen-binding portion thereof, (e.g.,adalimumab), may be administered to a human subject for treatment of adisorder associated with detrimental TNFa activity according to amonthly dosing schedule. In one embodiment, 80 mg of a human TNF alphaantibody, or antigen-binding portion thereof, in the formulation of theinvention (e.g., 0.8 mL of a 100 mg/mL formulation of the invention) isadministered to a subject having a disorder associated with detrimentalTNFa activity. In one embodiment, 80 mg of a human TNF alpha antibody,or antigen-binding portion thereof, in the formulation of the invention(e.g., 0.8 mL of a 100 mg/mL formulation of the invention) isadministered monthly or biweekly to a subject for the treatment of adisorder associated with detrimental TNFa activity.

Dose amounts described herein may be delivered as a single dose (e.g., asingle dose of 40 mg in 0.4 mL or 80 mg dose in 0.8 mL), or,alternatively may be delivered as multiple doses (e.g., four 40 mg dosesor two 80 mg doses for delivery of a 160 mg dose).

The formulation of the invention comprising an isolated human TNF alphaantibody, or antigen-binding portion thereof, (e.g., adalimumab) mayalso be administered to a subject in combination with an additionaltherapeutic agent. In one embodiment, the formulation is administered toa human subject for treatment of rheumatoid arthritis in combinationwith methotrexate or other disease-modifying anti-rheumatic drugs(DMARDs). In another embodiment, the formulation is administered to ahuman subject for treatment of JIA in combination with methotrexate orother disease-modifying anti-rheumatic drugs (DMARDs). Additionalcombination therapies are described in U.S. Pat. Nos. 6,258,562 and7,541,031; and U.S. Patent Publication No. US20040126372, the entirecontents of all of which are incorporated by reference herein.

The formulation of the invention comprising a human TNF alpha antibody,or antigen-binding portion thereof, may also be used to treat a subjectwho has failed previous TNF inhibitor therapy, e.g., a subject who haslost response to or is intolerant to infliximab.

The invention is further illustrated in the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1 High Concentration Anti-TNFα Antibody FormulationReduces Injection Pain

There have been reports of pain associated with the subcutaneousadministration of a human anti-TNFα antibody, e.g., adalimumab. Inplacebo-controlled trials, 20% of patients treated with adalimumabdeveloped injection site reactions (erythema and/or itching,haemorrhage, pain or swelling), compared to 14% of patients receivingplacebo. Most injection site reactions are mild and do not generallynecessitate drug discontinuation.

There are two main components of the injection pain associated withadalimumab: the pain associated with the needle stick, and the painassociated with the injection of drug into the tissue. Theinjection-related pain may be related to the Adalimumab formulationand/or to the volume of medication. The following study examined whethervarious formulations have an impact on injection pain followingsubcutaneous delivery of adalimumab.

Materials and Methods Study Design

The primary objectives of this study were to compare injection-relatedpain of three high concentration (100 mg/mL) adalimumab formulations inthe PHYSIOLIS™ pre-filled syringe with the current (50 mg/mL) adalimumabcommercial formulation in the current pre-filled syringe; and to assessthe bioavailability of three high concentration (100 mg/mL) adalimumabformulations in comparison to the current (50 mg/mL) adalimumabcommercial formulation. The secondary objective of this study was toassess the safety and tolerability of all four adalimumab formulations.

200 healthy adult male and female subjects who fulfilled the studyeligibility criteria were recruited to participate in the study.Generally, the study was conducted according to a randomizedparallel-group design. Pain assessment data was preferably obtained fromall 200 subjects. Evaluation of pharmacokinetics (PK) was done only forthe first 100 or so subjects.

Subjects from each treatment group were scheduled to receive onesubcutaneous injection of adalimumab 40 mg via a pre-filled syringe.There were four treatment groups, one for each of the four formulationsas set forth in Table 1 below. After meeting the selection criteria,subjects were randomly assigned in roughly equal numbers to one of thefour treatment groups shown in Table 1.

The three high concentration formulations (F1, F3, and F4), eachcontained 40 mg of adalimumab in 0.4 mL of solution in the PHYSIOLIS™pre-filled syringe. F1, F3, and F4 were compared to the currentadalimumab commercial formulation of 40 mg of adalimumab in 0.8 mL ofsolution in the current pre-filled syringe. The ingredients for each ofthe formulations is described below in Table 1. The formulationsdescribed in Table 1 also refer to the formulations described inExamples 2-7 below.

TABLE 1 Treatment Groups Treatment No. of Study Day 1 SC Group SubjectsInjection Formulation A 50 High Conc. adalimumab, Formulation 1 (F1)Mannitol, (40 mg/0.4 mL in Citric acid monohydrate, the PHYSIOLIS ™Sodium citrate, pre-filled syringe) Disodium phosphate dihydrate,Polysorbate 80, Water for injection, Sodium Hydroxide added as necessaryto adjust pH. B 50 High Conc. adalimumab, Formulation 3 (F3) Mannitol,(40 mg/0.4 mL in Polysorbate 80, the PHYSIOLIS ™ Water for injectionpre-filled syringe) C 50 High Conc. adalimumab, Formulation 4 (F4)Polysorbate 80, (40 mg/0.4 mL in Water for injection the PHYSIOLIS ™pre-filled syringe) D 50 Current adalimumab, Commercial Mannitol,Formulation Citric acid monohydrate, (40 mg/0.8 mL in Sodium citrate,the current pre-filled Disodium phosphate dihydrate, syringe) Sodiumdihydrogen phosphate dihydrate, Sodium chloride, Polysorbate 80, Waterfor injection, Sodium Hydroxide added as necessary to adjust pH

The first about 100 subjects to fulfill all entry criteria and enroll inthe study were randomized to the four treatment groups in roughly equalnumbers in each group and participated as either Cohort 1 or Cohort 2.The second about 100 subjects to fulfill all entry criteria and enrollin the study were randomized to the four treatment groups, in roughlyequal numbers in each group, and participated as Cohorts 3-5. It is theCohort number that specifies if a subject has pharmacokinetic (PK) andpain assessments or only pain assessments as described in Table 2 below.

TABLE 2 Assignment of Study Subjects Total No. of Subjects Cohort NAssessments A B C D 1 50 PK and Pain 13 12 13 12 2 44 PK and Pain 11 1210 11 3 38 Pain 9 9 10 10 4 39 Pain 10 10 10 9 5 29 Pain 7 7 7 8

Pharmacokinetic sample collection and pain assessments was done for allsubjects in the first two cohorts of about 100 patients (Cohorts 1 and2). Subjects in Cohorts 3-5 only participated in pain assessments, andno pharmacokinetic samples were be collected for these subjects. Safetyand tolerability were to be assessed in all subjects of all 5 cohorts.Each subject was randomly assigned to receive one injection ofadalimumab on Study Day 1. Each dose of study drug was to beadministered subcutaneously by an appropriate site staff member via apre-filled syringe in accordance with the proper injection method. Theinjection was given subcutaneously in the abdomen 2 inches to the rightof the navel. Questionnaires were administered by a different studystaff member than the individual administering the injection, as oftenas possible.

Subjects in Cohorts 1 and 2 (pharmacokinetic and pain assessments) wereconfined to the study site and supervised for approximately 10 days (9nights). Confinement for each subject began on Study Day −1 (1 day priorto the dosing day) and ended after the collection of the 192 hour bloodsamples and scheduled study procedures on Study Day 9. Serial bloodsamples were collected through Study Day 57 after dosing with subjectsreturning for outpatient visits. Safety and tolerability were assessedthroughout the study. Subjects in Cohorts 3-5 (pain assessments only)were confined to the study site and supervised for approximately 3 days(2 nights). Confinement for each subject began on Study Day −1 (1 dayprior to the dosing day) and ended after the completed study procedureson Study Day 2. Safety and tolerability were assessed throughout thestudy.

In addition to bioavailability and AAA assays, tolerability waspreferably assessed as follows:

1) Immediately following the injection on Study Day 1: Pain AssessmentModule was completed by the subject.

2) Approximately 10 minutes following the injection on Study Day 1:Draize Scale (hemorrhage, petechiae, erythema, edema, and pruritus) wasevaluated by a qualified site staff member.

3) Approximately 15 minutes following the injection on Study Day 1: PainAssessment Module was completed by the subject.

4) Approximately 30 minutes following the injection on Study Day 1: PainAssessment Module and Draize Scale evaluation was completed by thesubject and a qualified site staff member, respectively.

The demographics of subjects in the treatment groups are as follows,shown in Table 3, below.

TABLE 3 Patient Demographics High Conc. High Conc. High Conc. CommercialVar- Formulation 1 Formulation 3 Formulation 4 Formulation iable (N =50) (N = 50) (N = 50) (N = 50) Age 29.6 ± 8.7  29.5 ± 9.4 30.0 ± 8.930.3 ± 9.7  (yrs) Weight 68.3 ± 13.9 69.6 ± 9.6 67.0 ± 8.4 68.5 ± 10.0(kg) Sex 31 F (62%), 25 F (50%), 31 F (62%), 30 F (60%), 19 M (38%) 25 M(50%) 19 M (38%) 20 M (40%) Race 37 white 45 white 36 white 40 white(74%), (90%), (72%), (80%), 9 black (18%), 3 black (6%), 10 black (20%),8 black (16%), 4 other (8%) 2 other (4%) 4 other (8%) 2 other (4%)

Formulations

Three new high concentration formulations (referred to herein asFormulation 1, 3, or 4; or F1, F3, or F4, respectively) were studiedrelative to the commercial 50 mg/mL adalimumab formulation. Thecompositions of each of these formulations are listed below in Tables4-7.

TABLE 4 Formulation 1 (F1) COMPOSITION OF THE BULK SOLUTION 1 mL bulksolution contains Concentration Name of ingredient [mg] Active Substanceadalimumab (A-765865)*   100.00 Excipients Mannitol    42.00 Citric acidmonohydrate    1.31 Sodium citrate    0.31 Disodium phosphate dihydrate   1.53 Sodium dihydrogen phosphate dihydrate    0.86 Polysorbate 80   1.00 Sodium Hydroxide q.s. Water for injections ad 1,041.00 Nitrogen— Density of the solution: 1.041 g/mL *Used as concentrate.

TABLE 5 Formulation 3 (F3) COMPOSITION OF THE BULK SOLUTION 1 mL bulksolution contains Concentration Name of ingredientent [mg] ActiveSubstance adalimumab (A-765865)*  100  Excipients Mannitol    42.00Polysorbate 80    1.00 Water for injections ad 1,040.00 Nitrogen — *Usedas concentrate. Density of the solution: 1.040 g/mL

TABLE 6 Formulation 4 (F4) COMPOSITION OF THE BULK SOLUTION 1 mL bulksolution contains Concentration Name of ingredient [mg] Active Substanceadalimumab (A-765865)*   100.00 Excipients Polysorbate 80    1.00 Waterfor injections ad 1,026.00 Nitrogen — *Used as concentrate. Density ofthe solution: 1.026 g/mL

TABLE 7 Commercial 50 mg/mL adalimumab formulation COMPOSITION OF THEBULK SOLUTION 1 mL bulk solution contains Concentration Name ofingredient [mg] Active Substance adalimumab (A-765865)*      50.00Excipients Mannitol      12.00 Citric acid monohydrate       1.30 Sodiumcitrate       0.30 Disodium phosphate dihydrate       1.53 Sodiumdihydrogen phosphate dihydrate       0.86 Polysorbate 80       1.00Sodium Hydroxide q.s. Water for injections ad about 1,000   SodiumChloride       6.16 Nitrogen —

Study Drug Administration

Study drug (adalimumab) in the various formulations was administered inthe morning at Hour 0 on Study Day 1. The four treatment groups are setforth in Table 1 above as Groups A, B, C and D. Subjects in eachtreatment group were subcutaneously injected with only a singleadalimumab formulation via a pre-filled syringe.

Two types of syringes were used in this study; the currentlycommercially available glass pre-filled syringe (“current pre-filledsyringe”) was used for the reference current adalimumab commercialformulation (40 mg of adalimumab in 0.8 mL of solution), and thePHYSIOLIS™ pre-filled syringe for the three high concentration testformulations (40 mg of adalimumab in 0.4 mL of solution). The PHYSIOLIS™pre-filled syringe has a 29 gauge needle (the current pre-filled syringehas a 27 gauge ½ inch length fixed needle), a latex-free needle shield,and a plunger stopper which is coated to minimize leachables.

Pain Testing: Pain Scale

The Pain Visual Analog Scale was used to quantitatively assess painsensation. The following instructions were followed to assess the PainVisual Analog Scale (VAS):

The pain scale was administered to the subject at three different timesafter the injection: immediately after the injection, at 15 minutesafter the injection, and at 30 minutes after the injection on StudyDay 1. The pain scale was shown and read to the subject, who was askedto place one straight vertical mark along the line in the pain scale toindicate their current level of pain at the injection site (for examplesee below). An indication of 0 meant no pain, while the highest score(10) indicated “the worst imaginable pain.” An illustrative pain scaleused in the study is shown below:

What is your current level of pain at the injection site?

-   0 10-   no pain the worst imaginable pain

Qualitative Assessment of Pain

Following the completion of the pain scale, the qualitative assessmentof pain was administered three times after the injection: immediatelyfollowing the injection, at 15 minutes post the injection, and 30minutes following the injection on Study Day 1. An exemplary qualitativeassessment of pain used in the study is shown below:

Select all that describe your current level of pain at the injectionsite:Shooting painSharp pain

Stinging Pain Dull Uncomfortable Pressure Aching Soreness

Localized burning

Other OR

I currently have no discomfort at my injection site

Needle Pain Assessment

After the qualitative assessment of pain was completed, a needle painassessment was administered immediately following the injection. Anexemplary needle pain assessment used in the study is shown below:

Were you able to tell the difference between the pain from the needleentering your skin and the pain from the solution that was injected?

Yes No

a. If yes, was most of your pain caused by the needle entering your skinor was most of your pain caused by the solution that was injected?Most of my pain was caused by the needle entryMost of my pain was caused by the solution that was injected

Draize Scale

Qualified site personnel completed this assessment for each subjectapproximately 10 minutes and 30 minutes after the injection on Study Day1.

Hemorrhage/Petechiae at injection site:

0: None

1: Isolated; up to 5 petechiae

2: Isolated but >5 petechiae

3: Many petechiae, some coalescence

4: Spots of blood on surface

5: Frank bleeding

Erythema at injection site:

0: No erythema

1: Very slight (barely perceptible) erythema

2: Well-defined erythema

3: Moderate to severe erythema

4: Severe erythema (beet redness)

Edema at injection site:

0: No edema

1: Very slight (barely perceptible) edema

2: Slight edema (edges of area well defined by slight raising)

3: Moderate edema (raised ˜1 mm)

4: Severe edema (raised >1 mm, extending beyond area of injection)

Pruritis at injection site:

0: No pruritis

1: Occasional pruritis

2: Constant pruritis

Results

To determine whether delivery of adalimumab could be improved, new highconcentration formulations were developed. Formulations F1, F3, and F4,as shown below, have half of the volume (i.e., 0.4 mL vs. 0.8 mL) andtwice the protein concentration (100 mg/mL vs. 50 mg/mL) compared to thecurrent commercial adalimumab formulation, and they also have differentexcipient compositions. Experiments described herein were designed toassess whether any of the new formulations are superior to the currentcommercial adalimumab formulation.

The pain visual analog scale was chosen to assess injection site pain,and was used to evaluate impact of formulation composition on painsensations. In addition, tolerability of various new adalimumab 100mg/mL formulations were compared to the current commercial formulation(50 mg/mL adalimumab formulation). Data in this example supports thesurprising finding that the new formulations, especially Formulation 3(F3), decreases pain significantly relative to the current commercialformulation. Surprisingly, F3 also decreased pain significantly relativeto formulations F1 and F4.

Specifically, FIG. 1 shows that administration of high concentrationFormulations 1 and 3 resulted in a significant decrease in painassessment at all time points after injection (immediately, 15 minutes,and 30 minutes), compared to the other treatment groups (F4 and thecurrent commercial formulation). Table 8, shown below, summarizes theindividual data and shows a comparison of the F1, F3, and F4formulations with the 0.8 mL, 50 mg/mL commercial formulation.

As described in Table 8, immediately after injection, subjects whoreceived the current Humira formulation reported a mean (SD) pain scoreof 3.29 (2.57) cm. The mean pain scores for Formulation 1 andFormulation 3 were statistically significantly lower than that for thecurrent Humira formulation (p<0.001). The estimated differences from thecurrent Humira formulation were −1.50 (95% CI: −2.31-−0.69 cm) forFormulation F1, and −2.70 (95% CI: −3.52-−1.89 cm) for Formulation F3.Thus, Treatments A and B (high concentration Formulations 1 and 3)resulted in 45.6% and 82.7% reductions in injection site pain,respectively. Statistical tests were not performed for the pain scoresassessed at 15 minutes and 30 minutes after injection because a majorityof the subjects reported no pain at these time points. As described inFIG. 1, the minimum/maximum VAS scores immediately after injection wereas follows: Formulation F1, 0.00-8.3; Formulation F3, 0.00-2.20;Formulation F4, 0.20-8.70; and current commercial formulation,0.00-10.00.

It was apparent that the pain associated with the injection ofFormulation 3 was dramatically reduced compared to the same for thecurrent commercial formulation. Specifically, mean pain value, asassessed by the pain visual analog scale (VAS) immediately afterinjection, decreased from a mean of 3.29 in the current commercialformulation to 0.56 in Formulation 3, a stark 83% reduction. In fact,the pain reduction associated with Formulation 3 was so significant, itwas 69% reduced from the level of the next best formulation (in terms ofpain)-Formulation 1 (1.79).

Similarly, the mean pain scale for Formulation 1 reduced to 1.79, a 45%reduction from the 3.29 pain scale associated with the currentcommercial formulation.

TABLE 8 Pain Visual Analog Scales (VAS) Immediately After InjectionComparisons with Current Mean LS Formulation& Treatment N (SD) Mean&Estimate P-value# 95% CI High Conc. 50 1.79 1.79 −1.50 <0.001 −2.31,Formulation 1 (2.08) −0.69 High Conc. 50 0.56 0.58 −2.71 <0.001 −3.52,Formulation 3 (0.56) −1.90 High Conc. 50 4.12 4.11 0.82 0.976 0.01,Formulation 4 (2.50) 1.63 Current 50 3.29 3.29 Formulation (2.57) #Froma one-sided test with the null hypothesis being that the mean for thetest formulation is ≧ the mean for the current formulation &Based onANOVA.

A Qualitative Assessment of Pain was also administered to subjectsimmediately after injection, 15 minutes after injection and at 30minutes after injection for all four adalimumab treatments Immediatelyafter injection, an assessment of “no discomfort” was reported withgreatest frequency by 31 subjects (31/50, 62.0%) who had receivedFormulation 3, followed by 19 subjects (19/50, 38.0%) who had receivedFormulation 1, 7 subjects (7/50, 14.0%) who had received the currentHumira formulation, and one subject (1/50, 2.0%) who had receivedFormulation 4. Of those subjects who reported discomfort immediatelyafter injection, “stinging pain” was the most frequently reportedsensation with 30 subjects (30/50, 60%) for each of the currentformulation and formulation 4, 16 subjects (16/50, 32.0%) forFormulation 1, and 4 subjects (4/50, 8.0%) for Formulation 3. At 15minutes after injection, a large majority of subjects receiving eachtreatment reported “no discomfort” at the injection site.

Study site staff also utilized the Draize Scale to evaluatehemorrhage/petechiae, erythema, edema and pruritis at the injection siteof each subject. Ten minutes after injection the majority of subjects inall treatment groups had no observed injection-site hemorrhages orpetechiae, edema or pruritis.

All four formulations were well tolerated during the study. A summary ofpreliminary adverse events (AE) data is shown below in Table 9.

TABLE 9 Preliminary Adverse Events (AE) High Conc. High Conc. High Conc.Current Formulation Formulation Formulation Formulation 1 (N = 50) 3 (N= 50) 4 (N = 50) (N = 50) Any AE 7 (14%) 7 (14%) 6 (12%) 3 (6%) Any AEat least 3 (6%) 3 (6%) 2 (4%) 1 (2%) possibly drug related Any severe AE0 0 0 0 Any serious AE 0 0 0 0 Any AE leading 0 0 0 0 to studydiscontinuation Deaths 0 0 0 0

Accordingly, the data demonstrates that the new 100 mg/mL formulations,especially formulations 1 and 3, are well tolerated, and were effectivein reducing injection site pain after subcutaneous injection of similartherapeutic doses as compared to the currently marketed adalimumabformulation. Formulation F3 had a particularly low VAS score relative tothe other formulations tested.

The reduction in pain using the VAS score was not related to thedifference in needle size (a 27 G needle was used to administer thecurrent adalimumab commercial formulation and a 29 G needle was used toadminister formulations F1, F3, and F4). In particular, a needle prickaccounts for an immediate pain response, whereas the pain responsemeasured by the VAS scale indicated a prolonged persistent pain overseveral minutes, demonstrating that the injected solution itselfcontributes to the majority of the response. In addition, all of thetest formulations (F1, F3, and F4) were injected using the same sizeneedle, yet F1, F3, and F4 had very different VAS scores. This resultfurther demonstrates that it was the formulation contributing to thepain effect. and that this can be separated from the size of the needleused to administer the formulations.

Example 2 High Concentration Anti-TNFA Antibody Formulations IncreaseBioavailability in Humans

The following example describes a Phase 1, single-blind, single-dose,parallel-group design, randomized study in healthy volunteers (samestudy described above in Example 1). The primary objectives of thisstudy were to compare injection-related pain of three high concentration(100 mg/mL) adalimumab formulations in the Physiolis PFS with thecurrent (50 mg/mL) adalimumab (Humira) formulation in the current PFS(see Example 1), and to assess the bioavailability of three highconcentration (100 mg/mL) adalimumab formulations in comparison to thecurrent commercial (50 mg/mL) adalimumab (Humira) formulation. Thesecondary objective of this study was to assess the safety andtolerability of all four adalimumab formulations.

Study Design

Two hundred healthy volunteers were enrolled in this study (Table 10).Pain assessment data were obtained from all 200 subjects. Adalimumabpharmacokinetics were evaluated in the first 100 subjects. A descriptionof the formulations is provided above in Table 1.

TABLE 10 Treatment Groups Number of Number Subjects of for TreatmentSubjects Pharmacokinetic Study Day 1 Group for Study Data SC Injection A50 24 High Concentration Adalimumab Formulation No. 1 (40 mg/0.4 mL inthe Physiolis PFS) B 50 24 High Concentration Adalimumab Formulation No.3 (40 mg/0.4 mL in the Physiolis PFS) C 50 23 High ConcentrationAdalimumab Formulation No. 4 (40 mg/0.4 mL in the Physiolis PFS) D 50 23Current Commercial Humira Formulation (40 mg/0.8 mL in the current PFS)*See Tables 4-7 for formulation compositions.

Subjects from each treatment group received one subcutaneous injectionof 40 mg adalimumab via PFS on Study Day 1. Each dose of study drug wasadministered subcutaneously by an appropriate site staff member inaccordance with the proper injection method as described in theprotocol. The injection was given subcutaneously in the abdomen 2 inchesto the right of the navel. Questionnaires were administered by adifferent study staff member than the individual administering theinjection, as often as possible.

Results Pharmacokinetic Results and Conclusions

Following a single subcutaneous dose of adalimumab, the central valuesof the pharmacokinetic parameters, T_(max), C_(max), AUC₀₋₃₆₀ andAUC₀₋₁₃₄₄ were similar between Treatments A, B (high concentrationadalimumab Formulations 1 and 3, respectively) and D (current commercialHumira formulation). Following the single dose administration, meanT_(max) was earlier for Treatment C (high concentration adalimumabFormulation 4) relative to Treatment D (FIGS. 2 and 3). The centralvalues of C_(max) and AUC₀₋₃₆₀ values were greater (p<0.05) forTreatment C versus Treatment D.

Bioavailability/Bioequivalence for Treatments A, B and C (AdalimumabHigh Concentration Formulations) Relative to Treatment D (CommercialHumira Formulation)

For Treatment Group A versus D, the point estimates for the ratios ofC_(max), AUC₀₋₃₆₀, and AUC₀₋₁₃₄₄ central values for Treatments A and Bwere near unity, and the 90% confidence intervals were within the 0.80to 1.25 range. For Treatment B versus D, the point estimates for theratios of C. and AUC₀₋₃₆₀ central values were near unity and the 90%confidence intervals were within the 0.80 to 1.25 range. For AUC₀₋₁₃₄₄,the upper bound of the 90% confidence interval for Treatments B versus Dwas above 1.25. For Treatment C versus D, the point estimates for theratio of C_(max), AUC₀₋₃₆₀ and AUC₀₋₁₃₄₄ central values were 1.429,1.309, and 1.170 respectively, indicating that the relativebioavailability of Treatment C (Formulation 4) was greater.

TABLE 11 Relative Bioavailability and 90% Confidence Intervals for theBioequivalence Assessment Treat- Relative Bioavailability ments^(£) 90%Test vs. PK Central Value Point Confidence Reference Parameter TestReference Estimate Interval A vs. D C_(max) 4.47 4.39 1.018 0.859-1.207AUC₀₋₃₆₀ 1192.14 1192.23 1.000 0.860-1.163 AUC₀₋₁₃₄₄ 2306.91 2387.280.966 0.814-1.147 B vs. D C_(max) 4.52 4.39 1.029 0.868-1.219 AUC₀₋₃₆₀1222.24 1192.23 1.025 0.882-1.192 AUC₀₋₁₃₄₄ 2547.95 2387.28 1.0670.899-1.266 C vs. D C_(max) 6.28 4.39 1.429 1.202-1.699 AUC₀₋₃₆₀ 1561.051192.23 1.309 1.123-1.527 AUC₀₋₁₃₄₄ 2794.29 2387.28 1.170 0.983-1.394^(£)Treatments A, B or C: a single dose of high concentration adalimumabFormulation 1, 3 or 4, respectively, administered as a single scinjection using a Physiolis PFS (40 mg/0.4 mL). Treatment D: a singledose of the current Humira formulation administered as a single scinjection using the currently available glass PFS (40 mg/0.8 mL). PK =Pharmacokinetic.

Pharmacokinetic Conclusions

Based on the pharmacokinetic results, the relative bioavailability ofTreatments A and B were similar to Treatment D, the currently marketedHumira formulation. The relative bioavailability of Treatment C wasgreater when compared to Treatment D. The unexpected increase inbioavailability for Treatment C suggests that the effective dose amountadministered to a subject may be reduced.

Immunogenicity Conclusions

Twelve subjects had positive AAA samples during any time in the study,with only two subjects determined as AAA positive according to the predefined definition. Because of the small sample size and relativelysimilar numbers of AAA positive samples, no conclusions can be made ofthe immunogenicity between the treatments.

Safety Conclusions

The treatments tested were generally well tolerated by the subjects. Noclinically significant vital signs, ECG or laboratory measurements wereobserved during the course of the study. The majority of adverse eventswere assessed by the investigator as probably not or not related tostudy drug and mild in severity. No adverse events were assessed assevere.

No deaths, serious adverse events or discontinuations due to adverseevents occurred during the study.

Results of other safety analyses, including individual subject changesand potentially clinically significant values for vital signs, ECG andlaboratory measurements, were unremarkable for all treatment groups.

Tolerability

The tolerability assessments that were conducted included completion ofa Pain Assessment Module (Pain Visual Analog Scale [VAS]), QualitativeAssessment of Pain and Needle Pain Assessment) and the Draize Scale (seeExample 1).

Example 3 High Concentration Anti-TNFA Antibody Formulation IncreaseBioavailability in Pre-Clinical Model

The objective of the following study was to evaluate the pharmacokineticprofiles of adalimumab formulation F4 in contrast to the adalimumabcommercial formulation (see Table 7 above for a description of theformulation).

The pharmacokinetic profiles of HUMIRA (Adalimumab) were studied in maleand female Beagle dogs (2/sex/s.c. administration and 2 males/i.v.administration, Marshall Bio Resources USA, Inc., North Rose, N.Y.14516) after a single subcutaneous (s.c.) injection of the HUMIRAcommercial formulation (adalimumab) and a HUMIRA test formulationcorresponding to formulation F4 of the previous examples (adalimumab),as well as an intravenous (i.v.) injection of the HUMIRA commercialformulation as a control. The administered dose was 40 mg/dog (at 100mg/mL for F4 and 50 mg/mL of the commercial formulation).

For the determination of Adalimumab serum exposure levels, blood sampleswere collected at 0.083, 4, 24, 48, 96, 168, 240, 312, 384, 456, 528 and864 hours post administration (p.a.). Examined parameters were clinicalsigns (twice weekly) and mortality.

Apart from mucous feces in one male animal of the control group, norelevant clinical signs were observed. The incidences of clinical signsare summarized in Tables 14 and 15 below.

The pharmacokinetic results (described in Table 12 below) of this studyindicated that the bioavailability after s.c. dosing was about 80% andthe exposure levels seemed to be higher in females than in males afters.c. dosing. There was a trend for higher exposure levels following s.c.dosing of the test formulation compared with s.c. dosing of thecommercial formulation in males.

TABLE 12 Pharmacokinetic results AUC/Dose Animal AUC_(0-528 h)(μg*Hours/mL/ Vdss T_(1/2) Treatment Gender Number (μg*Hours/mL) mg/kg)(mL) (Hours) Test male 1001  9020 226 708 39.3 formulation, 1003 11400286 870 187.2  s.c. Mean 10200 ± 1680 256 ± 42.4 789 ± 115  113.3 ±104.6 female 1002 15400 384 388 55.5 1004 15800 395 469 54.5 Mean 15600± 283  390 ± 7.78 429 ± 57.3  55 ± 0.7 Commercial male 2001  8010 200692 21.4 formulation, 2003  8230 206 695 72.7 s.c. Mean 8120 ± 156 203 ±4.24 694 ± 2.12 47.1 ± 36.3 female 2002 12700 319 385 34.0 2004 17200431 477 119.0  Mean 15000 ± 3180 375 ± 79.2 431 ± 65.1 76.5 ± 60.1Commercial male 3001  9360 234 548 45.5 formulation, 3003 11900 298 40722.2 i.v. Mean 10600 ± 1800 266 ± 45.3 478 ± 99.7 33.9 ± 16.5

TABLE 13 Animal identification Animal No. Tattoo-No. Sex 1001 1246730Male 1003 1230230 Male 1002 1282302 Female 1004 1288688 Female 20011284879 Male 2003 1298951 Male 2002 1297237 Female 2004 1280491 Female3001 1285514 Male 3003 1290143 Male

TABLE 14 Summary of Clinical Observations in Males Dosage Group: 2 3 1Animals Examined: 4 4 2 Number Normal: 3 3 2 Category, Observation a b ab Excretion, feces 1 4 0 0 Note: a = Number of animals with observationb = Number of days observation seen

TABLE 15 Summary of Clinical Observations in Females Dosage Group: 2 3 1Animals Examined: 2 2 2 Number Normal: 2 2 2 Category, Observation Note:a = Number of animals with observation b = Number of days observationseen

Example 4 Stability of High Concentration Anti-TNFA AntibodyFormulations Against Freeze/Thaw Stress

The following example compares the stability of high concentrationformulations F1, F3, and F4 with the commercial adalimumab formulation.Stability was examined using freeze/thaw tests.

Experimental Setup

High concentration human anti-TNFα antibody formulations were preparedas described in Example 1, Table 1 above.

The compounded solutions were sterile filtered and aliquoted in 8×30 mLPETG bottles at 20 mL, respectively. The solutions were practically freefrom particles in visual inspection.

The samples for T0 were directly placed into a 2-8° C. refrigerator. Theother bottles were put into the −80° C. cube to freeze.

The next day the bottles were thawed in water baths with a temperatureof 25° C. or 37° C., respectively.

The Freeze/Thaw cycles were repeated 5 times. At T0 (before anyfreeze-thaw cycles), T1 (after one freeze-thaw cycle), T3 (after threefreeze-thaw cycles) and T5 (after five freeze-thaw cycles) samples weretaken for analysis and stored in a 2-8° C. fridge.

-   -   n=1 per pullpoint from 4 samples    -   Sample volume: 20 mL    -   Freeze/Thaw: −80° C./25° C.+37° C.    -   Freeze/thaw cycles: 5

After the cycling the samples were analyzed in the lab using each of thefollowing measures: Optical appearance (at each time point); absorptionat 340 nm; subvisible particles (at GGDDA);Photon-correlation-spectroscopy (PCS); Size Exclusion Chromatography(SEC); and Ion Exchange Chromatography (IEC).

Subvisible Particles

The measurement of subvisible particles was made at the Klotz particlemeasurement device. The results are shown in Table 16.

TABLE 16 Counts of particles >=1 μm, >=10 μm, and >=25 μm Particles >=Timepoint Sample Temperature Charge 1 μm 10 μm 25 μm T0 HC F1 (25° C.)E161118001CL 9 1 0 T0 HC F1 (37° C.) E161118001CL 7 2 1 T1 HC F1 25° C.E161118001CL 3 0 0 T1 HC F1 37° C. E161118001CL 33 1 0 T3 HC F1 25° C.E161118001CL 3 0 0 T3 HC F1 37° C. E161118001CL 20 1 0 T5 HC F1 25° C.E161118001CL 4 0 0 T5 HC F1 37° C. E161118001CL 94 0 0 T0 HC F3 (25° C.)E161119001CL 6 3 1 T0 HC F3 (37° C.) E161119001CL 12 2 0 T1 HC F3 25° C.E161119001CL 4 1 0 T1 HC F3 37° C. E161119001CL 7 2 0 T3 HC F3 25° C.E161119001CL 3 1 0 T3 HC F3 37° C. E161119001CL 9 2 1 T5 HC F3 25° C.E161119001CL 7 0 0 T5 HC F3 37° C. E161119001CL 5 0 0 T0 HC F4 (25° C.)E161120001CL 5 1 1 T0 HC F4 (37° C.) E161120001CL 7 1 0 T1 HC F4 25° C.E161120001CL 6 1 0 T1 HC F4 37° C. E161120001CL 5 1 0 T3 HC F4 25° C.E161120001CL 12 1 1 T3 HC F4 37° C. E161120001CL 60 0 0 T5 HC F4 25° C.E161120001CL 13 0 0 T5 HC F4 37° C. E161120001CL 22 1 0 T0 commercial(25° C.) E161121001CL 464 2 1 T0 commercial (37° C.) E161121001CL 198 00 T1 commercial 25° C. E161121001CL 143 1 0 T1 commercial 37° C.E161121001CL 285 0 0 T3 commercial 25° C. E161121001CL 108 0 0 T3commercial 37° C. E161121001CL 224 0 0 T5 commercial 25° C. E161121001CL39 0 0 T5 commercial 37° C. E161121001CL 151 0 0

The >=1 μm particle data showed a clear trend to a higher particle loadin Humira commercial and high concentration (HC) F1 at T5, reflecting acharacteristic behavior of buffer salt or sodium chloride containingadalimumab formulations.

Sample name Sum Aggregates Monomer Sum Fragments T0, HC F1, 25° C. 0.4299.50 0.09 T0, HC F1, 37° C. 0.43 99.46 0.11 T0, HC F3, 25° C. 0.3999.54 0.07 T0, HC F3, 37° C. 0.41 99.50 0.09 T0, HC F4, 25° C. 0.4399.46 0.11 T0, HC F4, 37° C. 0.42 99.48 0.11 T0, commercial, 25° C. 0.3699.55 0.09 T0, commercial, 37° C. 0.35 99.56 0.09 T1, HC F1, 25° C. 0.4399.47 0.10 T1, HC F1, 37° C. 0.44 99.48 0.08 T1, HC F3, 25° C. 0.3899.53 0.09 T1, HC F3, 37° C. 0.37 99.54 0.09 T1, HC F4, 25° C. 0.4499.47 0.09 T1, HC F4, 37° C. 0.44 99.46 0.10 T1, commercial, 25° C. 0.3599.56 0.08 T1, commercial, 37° C. 0.35 99.56 0.10 T3, HC F1, 25° C. 0.4299.47 0.10 T3, HC F1, 37° C. 0.42 99.48 0.11 T3, HC F3, 25° C. 0.4099.48 0.12 T3, HC F3, 37° C. 0.40 99.52 0.08 T3, HC F4, 25° C. 0.4899.41 0.11 T3, HC F4, 37° C. 0.44 99.48 0.08 T3, commercial, 25° C. 0.3699.54 0.10 T3, commercial, 37° C. 0.34 99.55 0.11 T5, HC F1, 25° C. 0.4399.48 0.09 T5, HC F1, 37° C. 0.45 99.45 0.10 T5, HC F3, 25° C. 0.4199.48 0.11 T5, HC F3, 37° C. 0.39 99.48 0.13 T5, HC F4, 25° C. 0.4799.43 0.10 T5, HC F4, 37° C. 0.49 99.40 0.11 T5, commercial, 25° C. 0.3699.56 0.08 T5, commercial, 37° C. 0.40 99.47 0.13

Subvisible particle counts for >=10 μm and >=25 both were very low.Freeze/thaw cycling did not lead to an increased number of subvisibleparticles, indicating that the tested formulations had favorablestability.

Size Exclusion Chromatography (SEC)

The SEC results are shown in Table 17. Table 17 indicates thepercentages of SEC aggregates, monomers, and fragments in each of thesolutions at T0 (before any freeze-thaw cycles), T1 (after onefreeze-thaw cycle), T3 (after three freeze-thaw cycles) and T5 (afterfive freeze-thaw cycles). These results indicate that each offormulations 1, 3, and 4 show stabilities similar to that of thecommercial formulation.

Table 17: Percentages of aggregates, monomers, and fragments before andafter freeze-thaw cycles as assessed by SEC

Ion Exchange Chromatography (IEC)

Ion exchange chromatography did not reveal different sensitivity of thetested solutions. No significant degradation could be observed.

However, with increasing number of freeze/thaw cycles, the samples thatwere thawed at 25° C. showed a higher amount in the 2nd acidic regionafter 5 cycles.

IEC results are shown in Table 18.

TABLE 18 IEC measurements before and after freeze-thaw cycles Sum Lysin1.ac 2.ac Peak Sample name Peaks area area Lysin 0 Lysin 1 between Lysin2 T0, HC F1, 25° C. 86.74 2.09 10.51 68.63 16.72 0.99 0.40 T0, HC F3,25° C. 87.29 1.89 10.22 66.35 16.16 0.91 3.87 T0, HC F4, 25° C. 87.351.85 10.20 66.46 16.15 0.89 3.85 T0, commercial, 87.18 1.93 10.22 66.2516.12 0.94 3.88 25° C. T1, HC F1, 25° C. 87.17 1.98 10.18 66.24 16.110.94 3.87 T1, HC F3, 25° C. 87.30 1.81 10.24 66.39 16.13 0.90 3.88 T1,HC F4, 25° C. 87.21 1.88 10.25 66.31 16.11 0.90 3.88 T1, commercial,87.19 2.01 10.20 66.27 16.11 0.93 3.88 25° C. T3, HC F1, 25° C. 87.231.94 10.20 66.34 16.12 0.91 3.87 T3, HC F3, 25° C. 87.27 1.86 10.2566.37 16.14 0.88 3.88 T3, HC F4, 25° C. 87.26 1.82 10.27 66.34 16.150.88 3.88 T3, commercial, 87.20 1.88 10.28 66.29 16.11 0.91 3.89 25° C.T5, HC F1, 25° C. 87.39 1.74 10.21 66.43 16.18 0.89 3.88 T5, HC F3, 25°C. 87.27 1.79 10.32 66.42 16.15 0.84 3.86 T5, HC F4, 25° C. 87.33 1.6910.32 66.49 16.14 0.85 3.85 T5, commercial, 87.05 1.95 10.38 66.17 16.100.89 3.88 25° C. T0, HC F1, 37° C. 87.25 1.94 10.19 66.36 16.13 0.903.86 T0, HC F3, 37° C. 87.42 1.82 10.19 66.54 16.16 0.85 3.87 T0, HC F4,37° C. 87.38 1.89 10.11 66.52 16.14 0.86 3.86 T0, commercial, 87.25 1.8610.24 66.34 16.12 0.91 3.88 37° C. T1, HC F1, 37° C. 87.21 1.98 10.1766.27 16.11 0.95 3.88 T1, HC F3, 37° C. 87.32 1.91 10.12 66.40 16.130.90 3.88 T1, HC F4, 37° C. 87.27 1.98 10.13 66.36 16.14 0.91 3.86 T1,commercial, 87.28 1.97 10.14 66.34 16.12 0.93 3.88 37° C. T3, HC F1, 37°C. 87.27 1.95 10.15 66.36 16.11 0.94 3.86 T3, HC F3, 37° C. 87.18 2.0310.11 66.29 16.13 0.90 3.87 T3, HC F4, 37° C. 87.21 1.95 10.15 66.3516.09 0.92 3.85 T3, commercial, 87.31 1.95 10.21 66.50 16.09 0.91 3.8137° C. T5, HC F1, 37° C. 87.29 2.01 10.06 66.40 16.11 0.93 3.85 T5, HCF3, 37° C. 87.25 2.07 10.06 66.37 16.10 0.92 3.87 T5, HC F4, 37° C.87.28 2.04 10.02 66.42 16.11 0.93 3.83 T5, commercial, 87.53 1.91 10.0266.72 16.11 0.88 3.83 37° C.

Example 5 Stability of High Concentration Anti-TNFA AntibodyFormulations Against Stir Stress

The following example describes a study which examined the stability ofthe F1, F3, and F4 formulations using the stir-stress test. Eachformulation was tested in a range of pH levels.

Materials

-   -   Humira HC F1 pH 4.2        -   100 mg/mL pH 4.7            -   pH 5.7            -   pH 6.2    -   Humira HC F3 pH 4.2        -   100 mg/mL pH 4.7            -   pH 5.7            -   pH 6.2    -   Humira HC F4 pH 4.2        -   100 mg/mL pH 4.7            -   pH 5.7            -   pH 6.2

Procedure

The vials, stir bars, and stoppers were steam sterilized prior to use.

The stirring experiment was performed with the following experimentalset-up:

-   -   Protein solutions: Humira HC F1, F3, F4, each at pH 4.2, 4.7,        5.7, 6.2 100 mg/mL, Humira HC F3 pH 5.2 100 mg/mL, Humira from        Vetter 50 mg/mL,    -   5 mL filling volume per 6R vial    -   n=3→2× stirred (with 7×2 mm magnetic bar), 1 unstirred control        (without magnetic bar)    -   magnetic stirrer multipoint HP: 550 rpm    -   ambient temperature    -   sample pull: t=0, t=1 h, t=4 h, t=24 h, t=48 h

Three 6R vials were filled with 5 mL for each protein solution andclosed with stoppers. Two of them were equipped with a magnetic stirbar.

The vials were kept at 5° C. over night. The next morning the samples(one per protein solution, because in the beginning they were all thesame) were measured with the turbidity meter. The measured solutionswere filled back in the vials prior start of the experiment. After 1, 4,24, and 48 h samples were taken and the turbidity was determined.

The unstirred samples were only measured at the time points 0 and 48 h.

For Humira HC F3 pH 5.2 also subvisible particles were determined forall time points.

Results Turbidity

The turbidity results for samples subjected to stir stress for 0, 1, 4,24, or 48 hours, as well as a 48 hour unstirred control, are shown inTable 19.

TABLE 19 Turbidity (NTU) of samples subjected to stir stress sample 0 h1 h 4 h 24 h 48 h Commercial 20.90 23.90 31.20 98.05 176.00 AdalimumabHumira HC F3 pH 5.2 6.13 6.69 8.92 18.05 29.50 Humira HC F1 4.2 8.628.89 9.40 6.48 15.05 Humira HC F1 4.7 14.00 15.50 20.05 10.88 81.40Humira HC F1 5.7 30.70 33.25 36.40 23.40 100.40 Humira HC F1 6.2 38.0040.95 52.60 32.65 168.00 Humira HC F3 4.2 3.20 3.35 3.72 4.88 6.69Humira HC F3 4.7 4.81 5.20 6.09 9.54 18.70 Humira HC F3 5.7 8.75 10.0311.30 25.90 46.10 Humira HC F3 6.2 9.24 — 13.05 22.60 37.30 Humira HC F44.2 3.44 3.74 3.80 6.48 9.79 Humira HC F4 4.7 5.13 5.67 6.60 10.88 17.00Humira HC F4 5.7 9.23 10.15 12.50 23.40 32.20 Humira HC F4 6.2 10.3011.65 15.55 32.65 56.75

Increased pH correlated with increased turbidity for all testedformulations, both T0/unstirred and stirred samples. This effect wasmost pronounced for formulation 1. Also, formulation 1 showed thehighest increase of turbidity after 48 h at all pH values except 4.2.Formulation 3 and 4 showed similar behavior and turbidity values werecomparable at all time points.

Humira HC (100 mg/mL), F3, pH 5.2 showed only a slight increase of theturbidity over the time. In contrast, the commercial Humira solutionshowed both a significantly higher starting value and increase inturbidity over the time. Thus, formulation 3 showed lower turbidity thanthe commercial Humira formulation.

The stirred samples showed a higher turbidity compared with theunstirred controls. The turbidity of the unstirred controls remainedalmost constant in comparison to the 0 h samples, indicating thatrunning the experiment at room temperature did not bias the results.

Subvisible Particles

Table 20 shows the results for the numbers of subvisible particles.

TABLE 20 Counts of subvisible particles before and after stirring stresssubvisible particles Humira HC F3 pH 5.2 >=1 μm >=10 μm >=25 μm  0 h 1033 1  1 h 194 4 0  4 h 202 4 0 24 h 262 2 0 48 h 192 3 0 48 h unstirred80 1 0

Particles ≧1 μm

Stirring induced a slight increase in sub-visible particle counts ≧1 μm.The unstirred control was comparable to the 0 h sample.

Particles ≧10 μm

Stirring had no significant effect on the particle counts ≧10 μm. Theunstirred control was comparable to the 0 h sample.

Particles ≧25 μm

Stirring had no significant effect on the particle counts ≧25 μm. Theunstirred control was comparable to the 0 h sample.

Overall, the results of the experiments presented in Example 5 showedthat formulation 3, when subjected to stirring stress, was surprisinglystable compared with commercial Humira solution. Formulation 3 wasrobust to stirring stress according to the turbidity measure, andstirring of formulation 3 also had little or no effect on formation ofsubvisible particles.

Example 6 Long-Term Storage Stability of High Concentration Anti-TNFAAntibody

The following example describes a study which examined the long-termstorage stability (up to 24 months) of the F1, F3, and F4 formulations.

Formulations F1, F3, and F4 were tested prior to long term storage(Initial), and after 3, 6, 9, 12, 18, and 24 months of storage. Thefollowing storage conditions were used: (1) 5° C., (2) 25° C./60%relative humidity (R.H), and (3) 40° C./75% R.H. During storage, thesamples were packaged in a 1 ml pre-filled syringe (colorless, glasstype I, Ph.Eur.); BD Hypak Syringe BD 260 with a grey DB Hypak 4023/50Fluorotec stopper. The following measures were used to assess storagestability:particulate contamination: visible particles; clarity andopalescence; color of solution (visual); in vitro TNFα-neutralization;cation exchange chromatography (CEX-HPLC), size exclusion chromatography(SE-HPLC); particulate contamination—sub-visible particles; containerclosure integrity; pH; and microbial quality.

All formulations tested were stable under the tested storage conditionsof 2-8° C. for up

Results

The results for Formulation F1 are presented in Table 21.

TABLE 21 Stability Summary Report for Formulation F1 Storage Conditions[° C./% r.H.] Duration 25° C./ 40° C./ \\Test Item ComponentSpecification of Testing 5° C. 60% R.H. 75% R.H. Participate Visible NMT4.5 Initial 0.0 0.0 0.0 contamination: particles 3 months 0.0 0.0 0.0visible 6 months 0.0 0.1 0.0 particles 9 months 0.0 — — 12 months 0.0 —— 18 months 0.0 — — 24 months 0.0 — — Clarity and Assessment Not moreInitial <=RS III <=RS III <=RS III Opalescence opalescent 3 months <=RSIII <=RS III <=RS IV than 6 months <=RS III <=RS III <=RS IV reference 9months <=RS IV — — suspension 12 months <=RS III — — IV 18 months <=RSIII — — 24 months <=RS III — — Color of BY-Scale Report value Initial<=BY 7 <=BY 7 <=BY 7 solution 3 months <=BY 7 <=BY 7 <=BY 7 (visual) 6months <=BY 7 <=BY 7 <=BY 6 9 months <=BY 7 — — 12 months <=BY 7 — — 18months <=BY 7 — — 24 months <=BY 7 — — In vitro TNF- (cytotoxicity 80%to 125% Initial 99 99 99 Neutralization test) [%] of the 3 months 97 11097 neutralization 6 months 87 81 68 capacity of 9 months 88 — — thereference 12 months 110 — — standard 18 months 97 — — 24 months 111 — —Fiducial Limit NLT 64 Initial 96.1 96.1 96.1 of error 3 months 91.1104.7 93.2 (p = 0.95) 6 months 84.2 76.3 64.4 lower Limit [%] 9 months84.3 — — 12 months 105.2 — — 18 months 95.3 — — 24 months 108.9 — —Fiducial Limit NMT 156 Initial 101.3 101.3 101.3 of error 3 months 101.9115.5 100.1 (p = 0.95) 6 months 90.8 85.9 71.2 upper Limit [%] 9 months90.8 — — 12 months 114.2 — — 18 months 98.8 — — 24 months 113.5 — —Cation Exchange Sum of lysine NLT 75 Initial 86.8 86.8 86.8Chromatography variants [%] 3 months 86.2 74.7 26.0 (CEX-HPLC) 6 months85.9 65.0 11.9 9 months 85.2 — — 12 months 85.2 — — 18 months 84.1 — —24 months 83.9 — — Size exclusion Principal peak NLT 98 Initial 99.699.6 99.6 chromatography (monomer) [%] 3 months 99.5 99.0 96.4 (SE-HPLC)6 months 99.4 98.5 92.9 9 months 99.4 — — 12 months 99.4 — — 18 months99.3 — — 24 months 99.3 — — Particulate Particles >=10 μm NMT 6000Initial 11 11 11 contamination - [/container] 3 months 8 37 55Sub-visible 6 months 33 102 98 Particles* 9 months 32 — — 12 months 58 —— 18 months 44 — — 24 months 11 — — Particles >=25 μm NMT 600 Initial 00 0 [/container] 3 months 0 0 1 6 months 0 2 2 9 months 0 — — 12 months0 — — 18 months 1 — — 24 months 0 — — Container Assessment Tight InitialComplies Complies Complies Closure 6 months Complies Complies CompliesIntegrity 12 months Complies — — 18 months Complies — — 24 monthsComplies — — pH Single values 4.7 to 5.7 Initial 5.3 5.3 5.3 3 months5.2 5.2 5.2 6 months 5.3 5.3 5.3 9 months 5.3 — — 12 months 5.3 — — 18months 5.3 — — 24 months 5.3 — — Microbial Sterility drug No evidenceInitial Complies Complies Complies quality product of microbial growthis found

The results described above show that when stored for 24 months at 5°C., Formulation F1 showed no visible particulate contamination, clarityand opalescence <=RS III, and visual color <=BY7(brown yellow 7).Formulation F1 also demonstrated 111% of the TNF-neutralization capacityof the reference standard, 83.9% lysine variants, 99.3% monomers, 11particles >=10 μm, and no particles >=25 μm. Furthermore, F1 maintaineda stable pH of 5.3 and showed no evidence of microbial growth. Whenstored for 6 months at 25° C./60% R.H., Formulation F1 showed 0.1visible particles, clarity and opalescence <=RS III, visual color <=BY7,81% of the TNF-neutralization capacity of the reference standard, 65%lysine variants, 98.5% monomers, 102 particles >=10 μm, 2 particles >=25μm, a stable pH of 5.3 and no evidence of microbial growth. When storedfor 6 months at 45° C./75% R.H., Formulation F1 showed no visibleparticles, clarity and opalescence <=RS IV, visual color <=BY6, 68% ofthe TNF-neutralization capacity of the reference standard, 11.9% lysinevariants, 92.9% monomers, 98 particles >=10 μm, 2 particles >=25 μm, andno evidence of microbial growth.

The results for Formulation F3 are presented in Table 22.

TABLE 22 Stability Summary Report for Formulation F3 Storage Conditions[° C./% r.H.] Duration 25° C./ 40° C./ Test Item Component Specificationof Testing 5° C. 60% R.H. 75% R.H. Participate Visible NMT 4.5 Initial0.0 0.0 0.0 contamination: particles 3 months 0.0 0.0 0.2 visibleparticles 6 months 0.2 0.1 0.0 9 months 0.0 — — 12 months 0.0 — — 18months 0.0 — — 24 months 0.0 — — Clarity and Assessment Not more Initial<=RS II <=RS II <=RS II Opalescence opalescent 3 months <=RS II <=RS II<=RS II than 6 months <=RS II <=RS II <=RS II reference 9 months <=RS II— — suspension 12 months <=RS II — — IV 18 months <=RS II — — 24 months<=RS II — — Color of BY-Scale Report value Initial <=BY 7 <=BY 7 <=BY 7solution 3 months <=BY 7 <=BY 7 <=BY 7 (visual) 6 months <=BY 7 <=BY 7<=BY 6 9 months <=BY 7 — — 12 months <=BY 7 — — 18 months <=BY 7 — — 24months <=BY 7 — — In vitro TNF- (cytotoxicity 80% to 125% Initial 87 8787 Neutralization test) [%] of the 3 months 101 106 89 neutralization 6months 100 101 90 capacity of 9 months 98 — — the reference 12 months 96— — standard 18 months 96 — — 24 months 98 — — Fiducial Limit NLT 64Initial 85.4 85.4 85.4 of error 3 months 92.9 88.1 80.6 (p = 0.95) 6months 98.3 97.4 86.5 lower Limit [%] 9 months 97.0 — — 12 months 93.3 —— 18 months 93.9 — — 24 months 96.7 — — Fiducial Limit NMT 156 Initial88.5 88.5 88.5 of error 3 months 110.5 122.2 97.9 (p = 0.95) 6 months101.7 103.8 92.6 upper Limit [%] 9 months 99.8 — — 12 months 99.2 — — 18months 98.1 — — 24 months 99.6 — — Cation Exchange Sum of lysine NLT 75Initial 86.8 86.8 86.8 Chromatography variants [%] 3 months 86.6 77.832.8 (CEX-HPLC) 6 months 86.4 70.1 16.5 9 months 86.0 — — 12 months 86.2— — 18 months 85.2 — — 24 months 85.1 — — Size exclusion Principal peakNLT 98 Initial 99.7 99.7 99.7 chromatography (monomer) [%] 3 months 99.699.2 96.9 (SE-HPLC) 6 months 99.5 98.8 93.8 9 months 99.5 — — 12 months99.5 — — 18 months 99.4 — — 24 months 99.4 — — ParticipateParticles >=10 μm NMT 6000 Initial 10 10 10 contamination - [/container]3 months 12 45 73 Sub-visible 6 months 22 157 275 Particles* 9 months 50— — 12 months 54 — — 18 months 45 — — 24 months 14 — — Particles >=25 μmNMT 600 Initial 0 0 0 [/container] 3 months 0 0 1 6 months 0 2 9 9months 0 — — 12 months 1 — — 18 months 0 — — 24 months 0 — — ContainerAssessment Tight Initial Complies Complies Complies Closure 6 monthsComplies Complies Complies Integrity 24 months Complies — — pH Singlevalues 4.7 to 5.7 Initial 5.2 5.2 5.2 3 months 5.3 5.3 5.3 6 months 5.25.2 5.3 9 months 5.3 — — 12 months 5.4 — — 18 months 5.2 — — 24 months5.1 — — Microbial Sterility drug No evidence Initial Complies CompliesComplies quality product of microbial growth is found

The results provided in Table 22 indicate that when stored for 24 monthsat 5° C., Formulation F3 showed no visible particulate contamination,clarity and opalescence <=RS II, and visual color <=BY7. Formulation F3showed 98% of the TNF-neutralization capacity of the reference standard,85.1% lysine variants, 99.4% monomers, 14 particles >=10 μm, and noparticles >=25 μm. The pH showed little change and there was no evidenceof microbial growth.

When stored for 6 months at 25° C./60% R.H., Formulation F3 showed novisible particles, clarity and opalescence <=RS II, and visual color<=BY7. Also, formulation F3 showed 101% of the TNF-neutralizationcapacity of the reference standard, 97.4% lysine variants, 70.1%monomers, 157 particles >=10 μm, and 2 particles >=25 μm. The pH wasstable and there was no evidence of microbial growth.

When stored for 6 months at 45° C./75% R.H., Formulation F3 showed novisible particles, clarity and opalescence <=RS II, and visual color<=BY6. Also, formulation F3 showed 90% of the TNF-neutralizationcapacity of the reference standard, 16.5% lysine variants, 93.8%monomers, 275 particles >=10 μm, and 9 particles >=25 μm. The pH wasquite stable, and there was no evidence of microbial growth.

The results for Formulation F4 are presented in Table 23.

TABLE 23 Stability Summary Report for Formulation F4 Storage Conditions[° C./% r.H.] Duration 25° C./ 40° C./ Test Item Component Specificationof Testing 5° C. 60% R.H. 75% R.H. Appearance Visual NMT 4.5 Initial 0.00.0 0.0 particles 3 months 0.0 0.0 0.0 6 months 0.0 0.0 0.0 9 months 0.0— — 12 months 0.0 — — 18 months 0.0 — — Clarity Assessment Not moreInitial <=RS II <=RS II <=RS II opalescent 3 months <=RS II <=RS II <=RSII than 6 months <=RS II <=RS II <=RS II reference 9 months <=RS II — —suspension 12 months <=RS II — — IV 18 months <=RS II — — Color BY-ScaleInitial <=BY 7 <=BY 7 <=BY 7 3 months <=BY 6 <=BY 6 <=BY 6 6 months <=BY7 <=BY 7 <=BY 6 9 months <=BY 7 — — 12 months <=BY 7 — — 18 months <=BY7 — — In vitro TNF- (cytotoxicity 80% to 125% Initial 111 111 111Neutralisation test) [%] of the 3 months 105 101 80 (Cytotoxi-neutralization 6 months 97 101 76 zitatstest) capacity of 9 months 112 —— the reference 12 months 97 — — standard 18 months 104 — — FiducialLimit NLT 64 Initial 105.2 105.2 105.2 of error 3 months 103.2 100.179.2 (p = 0.95) 6 months 92.9 97.5 74.7 lower Limit 9 months 109.3 — —[%] 12 months 90.2 — — 18 months 101.2 — — Fiducial Limit NMT 156Initial 116.3 116.3 116.3 of error 3 months 106.2 102.7 80.4 (p = 0.95)6 months 101.1 103.5 78.1 upper Limit 9 months 113.9 — — [%] 12 months104.9 — — 18 months 107.5 — — Cation Exchange Sum of lysine NLT 75Initial 85.5 85.5 85.5 Chromatography variants [%] 3 months 85.8 76.831.6 (CEX-HPLC) 6 months 85.4 68.7 15.7 9 months 85.2 — — 12 months 84.5— — 18 months 84.4 — — Size exclusion Principal peak NLT 98 Initial 99.799.7 99.7 chromatography (monomer) [%] 3 months 99.6 99.1 96.5 (SE-HPLC)6 months 99.6 98.8 93.1 9 months 99.5 — — 12 months 99.5 — — 18 months99.4 — — Participate Particles >=10 μm NMT 6000 Initial 17 17 17contamination - [/container] 3 months 51 174 207 Sub-visible 6 months 39144 218 Particles 9 months 82 — — 12 months 57 — — Particles >=25 μm NMT600 Initial 0 0 0 [/container] 3 months 0 1 5 6 months 0 1 1 9 months 1— — 12 months 2 — — Container Assessment Must comply Initial CompliesComplies Complies closure (no blue 6 months Complies Complies CompliesIntegrity coloration) pH Single values 4.7 to 5.7 Initial 5.1 5.1 5.1 3months 5.2 5.2 5.1 6 months 5.2 5.1 5.2 9 months 5.2 — — 12 months 5.2 —— 18 months 5.1 — — Microbial Sterility drug No evidence InitialComplies Complies Complies quality product of microbial growth is found

The results provided in Table 23 indicate that when stored for 18 monthsat 5° C., Formulation F4 showed no visible particulate contamination,clarity and opalescence <=RS II, and visual color <=BY7. Formulation F4showed 104% of the TNF-neutralization capacity of the referencestandard, 84.4% lysine variants, and 99.4% monomers. Furthermore, the pHwas stable and there was no evidence of microbial growth.

When stored for 6 months at 25° C./60% R.H., Formulation F4 showed novisible particles, clarity and opalescence <=RS II, and visual color<=BY7. Formulation F4 showed 101% of the TNF-neutralization capacity ofthe reference standard, 68.7% lysine variants, 98.8% monomers, 144particles >=10 μm, and 1 particle >=25 μm. Furthermore, the pH was quitestable and there was no evidence of microbial growth.

When stored for 6 months at 45° C./75% R.H., Formulation F4 showed novisible particles, clarity and opalescence <=RS II, and visual color<=BY6. Formulation F4 showed 76% of the TNF-neutralization capacity ofthe reference standard, 15.7% lysine variants, 93.1% monomers, 218particles >=10 μm, and 1 particle >=25 μm. Furthermore, the pH was quitestable and there was no evidence of microbial growth.

In summary, the results of the long-term stability experiments, aspresented in Tables 21-23, show that high concentration formulations F1,F3, and F4 were surprisingly stable when subjected to long term storage.The stability of these formulations was similar to the commercialformulation. Formulations F1 and F3 showed stability similar to thecommercial formulation after long term storage for at least 24 months.Formulation F4 showed stability similar to the commercial formulationafter long term storage for at least 18 months.

Example 7 Room Temperature Storage Stability of High ConcentrationAnti-TNFA Antibody

Liquid pharmaceutical products containing therapeutic antibodies oftenrequire storage at 2-8° C. until end-of-shelf-life. Cooling is thereforealso required by patients between purchasing of the medicines until use.Depending on the proposed dosing regimen, this can result in storagetimes under patient's responsibility in the case of self-administrationdrugs for up to several weeks.

Therefore, drugs that do not require storage under refrigeratedconditions display both a significant increase in patient conveniencefor home care products and reduction of drug quality concerns in case ofimproper storage, thereby reducing complaint rates and temperatureexcursion investigations.

The currently marketed Adalimumab containing product (Humira) wassuccessfully reformulated at a higher protein concentration asFormulation F3, as described above in Examples 1-6. The followingstability data for Formulation F3 resulted in findings of improvedstability against protein degradation. The resulting degradationkinetics measured at 25° C. complied with requirements for ambientstorage for up to 3 months.

For general long-term stability data related to storage at 25° C. ofrFormulation F3, see Example 6 above.

The following data describes long-term storage characteristics forFormulation F3. The data shows that even after 18 months and 24 monthsof long-term storage at 2-5° C., additional storage at 25° C./30° C. isacceptable.

TABLE 24 24m storage of F3 at 2-8° C., followed by 7 days/14 days ataccelerated conditions (25° C., 30° C.) Test t0 +7 Days +14 Dayscriterion Specification Characteristic: (24M 5° C.) 25° C. 30° C./65%R.H. 25° C. 30° C./65% R.H. Appearance colourless to complies compliescomplies complies complies slightly yellow solution Visible single vial≦2=> 0.0 Value per analyst: Value per analyst: Value per analyst: Valueper analyst: particles* practically free 3 × 0; single 3 × 0; single 3 ×0; single 3 × 0; single from visible vial ≦2=> vial ≦2=> vial ≦2=> vial≦2=> particulate matter practically free practically free practicallyfree practically free 1 vial >10=> from visible from visible fromvisible from visible particulate matter particulate matter particulatematter particulate matter Size Purity % monomer aggregate 0.4 0.4 0.40.4 0.4 exclusion NLT 98% monomer 99.4 99.3 99.3 99.3 99.2 HPLCfragments 0.2 0.2 0.3 0.3 0.3 Cation NMT 8% first acid region 2.7 2.83.0 2.9 3.3 exchange NMT 16% second acid region 10.5 10.9 11.4 11.6 12.7HPLC NLT 75% sum Lysine variants 85.1 84.2 83.4 83.0 81.2 NMT 4% peakbetween Lysine 1 1.5 1.7 1.9 1.6 1.8 and Lysine 2 report value [%] peaksafter Lysine 2 0.2 0.4 0.4 0.9 1.0 PCS report value [%] Z-Average 1.3901.365 1.395 1.397 1.420 Pdl 0.193 0.176 0.188 0.175 0.210

TABLE 25 18m storage at 2-8° C. of F3, followed by 7 days/14 days ataccelerated conditions (25° C., 30° C.) Test t0 +7 Days +14 Dayscriterion Specification Characteristics (18M 5° C.) 25° C. 30° C./65%R.H. 25° C. 30° C./65% R.H. Appearance colourless to complies compliescomplies complies slightly yellow solution Visible single vial ≦2=>Value per analyst: Value per analyst: Value per analyst: Value peranalyst: particles* practically free 3 × 0; single 3 × 0; single 3 × 0;single 3 × 0; single from visible vial ≦2=> vial ≦2=> vial ≦2=> vial≦2=> particulate matter practically free practically free practicallyfree practically free 1 vial >10=> from visible from visible fromvisible from visible inform lab manager particulate matter particulatematter particulate matter particulate matter Participate particles ≦ 10 ≦1 μm/1 ml 2250 5623 9355 10252 contamination, μm: ≦6000 ≦10 μm/1 ml 67 39 45 subvisible particles/container ≦25 μm/1 ml 0 0 0 1 particles*particles ≦ 25 μm: ≦600 particles/container PCS report value [%]Z-Average 2.378 2.344 2.353 2.358 Pdl 0.102 0.077 0.077 0.077

Example 8 Conductivity of High Concentration Anti-TNFA AntibodyFormulations

The conductivity of the high concentration anti-TNFα antibodyformulations F3 and F4 (see Examples 1-6, supra) was determined using anInoLab Cond Level2 WTW device normalized to 25° C. Table 26 shows theinfluence of non-ionic excipients on the conductivity of the F3 and F4adalimumab formulations.

TABLE 26 Conductivity of Formulations F3 and F4 Temperature ConductivitySample [° C.] [μS/cm] Adalimumab DP F3 1 22.4 663 2 22.4 651 3 23.8 6604 21.4 715 5 21.7 691 6 23.1 680 7 23.3 644 8 22.9 647 Adalimumab DP F41 22.0 797 2 22.9 746

As described above in Table 26, average conductivity for bothformulations F3 and F4 was less than 2 mS/cm.

Example 9 Dynamic Light Scattering (DLS) of High Concentration Anti-TNFAAntibody Formulations

Dynamic light scattering analysis of diluted solutions was used toassess the hydrodynamic diameter (reported as the mean or Z-averagesize, calculated by cumulants analysis of the DLS measured intensityautocorrelation function and polydispersity index, PDI, of the sizedistribution of particles). DLS measurements were specifically used todetect low amounts of higher molecular weight species, e.g. aggregates,in a size distribution, since those species possess higher scatteringintensity (proportional to d6) and, therefore, will influence theZ-average and Polydispersity Index (PDI) as an indicator of theZ-average size distribution significantly.

A 150 μL sample of each of formulations F3 and F4 (see examples 1-6above) was measured to analyze the average size of the particles(Z-average) and the Polydispersity Index (PDI), an indicator of the“broadness” of the particle size distribution using DLS. The results areshown below. DLS data did not show any signs of the development ofhigher molecular weight aggregates, since the polydispersity index, asensitive indicator for low levels of higher molecular weightsub-populations did not increase significantly.

Formulation F3

Sample No. ZAve (nm) PDI 1 2.4 n. a. 2 2.3 0.08 3 2.3 0.14 4 2.3 0.09

Formulation F4

Sample No. ZAve (nm) PDI 1 1.3 n.a. 2 2.5 n.a.

As described above, the z-average measurement for both F3 and F4 wasless than 4 nm. This low hydrodynamic diameter is representative of thefact that both formulations F3 and F4 do not contain additionalexcipients other than a polysorbate and a polyol or a polysorbate.

Example 10 Factors Influencing the Stability of High ConcentrationAnti-TNFA Antibody Formulations

The effect of varying mannitol concentrations and polysorbateconcentrations on the stability of adalimumab in water was examined.

Formulations containing 100 mg/ml of adalimumab in water were prepared.Subsequently, various concentrations of either mannitol or polysorbatewere added in a concentration range to determine the impact of eachexcipient on the stability of the formulation, as measured byaggregation and fragmentation. The concentrations of polysorbate andmannitol ranged from 0.1 to 1.0 mg/ml and 0-72 mg/ml, respectively, asshown in FIGS. 3A and 3B. As shown in FIG. 3A, varying the concentrationof mannitol from about 12 to about 72 mg/ml had a minimal effect on thestability of adalimumab. Similarly, varying the concentration ofpolysorbate-80 from about 0.1 to about 1.0 mg/ml had no effect on thestability of adalimumab.

INCORPORATION BY REFERENCE

The contents of all cited references (including, for example, literaturereferences, patents, patent applications, and websites) that maybe citedthroughout this application are hereby expressly incorporated byreference in their entirety for any purpose. The practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques of protein formulations, which are well known in the art.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are therefore intended to be embracedherein.

What is claimed:
 1. A liquid aqueous formulation comprising: (1) anisolated human anti-TNFα antibody, or an antigen-binding portionthereof; (2) a surfactant; and, (3) less than 50 mg/ml of a polyol;wherein the formulation has a pH of 4.7 to 5.7 and does not contain abuffer or a salt, and wherein injection of the formulation into a humansubject results in a Pain Visual Analog Scale (VAS) score of less than1.0.
 2. The formulation of claim 1, wherein the polyol is mannitol orsorbitol.
 3. The formulation of claim 2, comprising about 38-46 mg/ml ofmannitol.
 4. The formulation of claim 1, wherein the surfactant is apolysorbate.
 5. The formulation of claim 1, wherein the concentration ofthe anti-TNFα antibody, or antigen-binding portion thereof, is 95-105mg/ml.
 6. The formulation of claim 1, wherein is the antibody, or anantigen-binding portion thereof, comprises a light chain variable region(LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution atposition 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 5, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 7; and a heavy chain variable region (HCVR)having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4,or modified from SEQ ID NO: 4 by a single alanine substitution atposition 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising theamino acid sequence of SEQ ID NO: 6, and a CDR1 domain comprising theamino acid sequence of SEQ ID NO:
 8. 7. The formulation of claim 1,wherein the antibody, or an antigen-binding portion thereof, comprises alight chain variable region (LCVR) comprising the amino acid sequence ofSEQ ID NO: 1 and a heavy chain variable region (HCVR) comprising theamino acid sequence of SEQ ID NO:
 2. 8. The formulation of claim 1,wherein the antibody, or an antigen-binding portion thereof, isadalimumab.
 9. A liquid aqueous formulation consisting essentially of(1) a concentration of 90-110 mg/ml of an isolated human anti-TNFαantibody, or an antigen-binding portion thereof, having a light chainvariable region (LCVR) having a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a singlealanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 5, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 7; and having a heavychain variable region (HCVR) having a CDR3 domain comprising the aminoacid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 8; (2) apolysorbate; and, (3) about 38-46 mg/ml of mannitol.
 10. The formulationof claim 9, wherein the antibody, or an antigen-binding portion thereof,comprises a light chain variable region (LCVR) comprising the amino acidsequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO:
 2. 11. The formulationof claim 9, wherein the antibody, or an antigen-binding portion thereof,is adalimumab.
 12. The formulation of claim 9, comprising about 30-90 mgof the antibody, or antigen binding portion thereof.
 13. A method oftreating a disorder associated with detrimental TNFα activity in apatient, comprising administering to the patient the formulation ofclaim
 1. 14. A method of improving the bioavailability of an isolatedhuman anti-TNFα antibody, or an antigen-binding portion thereof, in ahuman subject, said method comprising administering a formulationcomprising a surfactant and an effective amount of the antibody, orantigen-binding portion thereof, to the subject such that thebioavailability of the antibody, or antigen-binding portion thereof, isimproved, wherein the formulation does not contain a buffer, a polyol,or a salt.
 15. The method of claim 14, wherein the effective amount ofthe antibody, or antigen-binding portion thereof, is 30-90 mg.
 16. Themethod of claim 14, wherein the concentration of the antibody, orantigen-binding portion thereof, in the pharmaceutical formulation is90-110 mg/ml.
 17. The method of claim 14, wherein the bioavailability ofthe antibody, or antigen-binding portion thereof, is an AUC₀₋₃₆₀ greaterthan 1300 μg*hr/ml when subcutaneously injected into the human subject.18. The method of claim 14, wherein the surfactant is a polysorbate. 19.The method of claim 14, wherein is the antibody, or an antigen-bindingportion thereof, is selected from the group consisting of a) anantibody, or an antigen-binding portion thereof, comprising a lightchain variable region (LCVR) having a CDR3 domain comprising the aminoacid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a singlealanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 5, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 7; and a heavy chainvariable region (HCVR) having a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 8; b) anantibody, or an antigen-binding portion thereof, comprises a light chainvariable region (LCVR) comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region (HCVR) comprising the amino acidsequence of SEQ ID NO: 2; and c) adalimumab.
 20. A pre-filled syringe orautoinjector device, comprising the formulation of claim 1.